JP7012004B2 - Drugs, uses and methods for the treatment of synucleinopathy - Google Patents

Description

The present invention relates to novel types of monoclonal antibodies that specifically bind to α-synuclein, as well as methods of using these molecules and their α-synuclein binding fragments for the treatment and diagnosis of synucleinopathy.

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Synucleinopathy, also known as Lewy body disease (LBD), is an intracellular protein cluster seen microscopically as Lewy bodies (LB) and / or Lewy neurites, of which the protein α-synuclein is a major component. Characterized by the deposition of aggregates (Jellinger, Mov Disord. 2012 Jan; 27 (1): 8-30; McKeith et al., Neurology (1996) 47: 1113-24). Synucleopathies are also known as Parkinson's disease (PD) (including idiopathic and hereditary Parkinson's disease) and diffuse Lewy body (DLB) disease (Lewy body dementia (DLB)). Lewy body variant Alzheimer's disease (LBV), combined Alzheimer's disease and Parkinson's disease (CAPD), pure autonomic dysfunction (PAF) and multiple system atrophy (MSA; eg, Olive Bridge cerebral atrophy, striatum) Black degeneration and Shy-Drager syndrome)). Synucleopathies often have degeneration of the dopaminergic black streak system, which is responsible for the major motor disorders (constriction, slow movement, resting tremor) in Parkinson's disease, but the central nervous system, There is also widespread development of Levy bodies and degenerative Levy neuropathy in the peripheral and autonomic nervous system and brain regions and in other organs associated with non-motor dysfunction such as dementia and autonomic nervous system disorders. Some of the non-motor signs and symptoms are thought to precede motor symptoms in Parkinson's disease and other synucleinopathy. Such early signs include, for example, REM sleep behavior disorder (RBD) and decreased sense of smell and constipation (Mahowald et al., Neurology (2010) 75: 488-489). Synucleinopathy continues to be a common cause of movement disorders and cognitive decline in the aging population (Galasco et al., Arch. Neurol. (1994) 51: 888-95).

Alpha-synuclein is one of a family of proteins including β- and γ-synuclein and synuclein. Alpha-synuclein is normally expressed in relation to synapses and is thought to play a role in regulating synaptic vesicle release, thereby affecting neurotransmission, plasticity, learning and memory.

Several studies suggest that α-synuclein plays a central role in the development of PD. Proteins can aggregate to form intracellular insoluble fibrils in pathological conditions. For example, synucleins accumulate in LB (Spillantini et al., Nature (1997) 388: 839-40; Takeda et al., J. Pathol. (1998) 152: 367-72; Wakabayashi et al., Neurosci. Let. (1997) 239: 45-8). In rare familial Parkinson's disease, mutations in the α-synuclein gene and duplication and triplication of this gene co-separate (Kruger et al., Nature Gen. (1998) 18: 106-8; Polymeropoulos, et al., Science (1997) 276: 2045-7). There has been an important finding that α-synuclein is secreted into extracellular fluid and can be present in plasma and cerebrospinal fluid (CSF). For example, Pacheco et al. (2015) and others (Pacheco et al J Neurochem. 2015 Mar; 132 (6): 731-4; Conway et al., Proc Natl Acad Sci USA (2000) 97: 571-576; Volles et al., J. Mol. Several studies by Biochem. 42: 7871-7878, 2003) suggest that extracellular synucleins play a pathogenic role in the brain. These studies demonstrated that extracellular α-synuclein oligomers are neurotoxic to the brain nerve cell membrane. Another interesting hypothesis based on synuclein secretion data is that the prion-like spread of α-synuclein underlies the progression of Parkinson's disease and other synucleinopathy (Lee et al. 2014, Nat). Rev Neuro. 2014 Feb; 10 (2): 92-8; Hansen and Li 2012, Trends Mol Med. 2012 May; 18 (5): 248-55). These findings raised the expectation that extracellular synucleins could be targeted by immunotherapy (Vekrellis et al. 2011, Lancet Neurol. 2011 Nov; 10 (11): 1015-25).

Native α-sinucrane autoantibodies have been shown to be present in both PD patients and healthy controls (Smith et al. 2012, PLoS One. 2012; 7 (12): e52285; Matzler et al. 2014, PLoS. One. 2014 Feb 21; 9 (2): e88604, Papachroni et al. 2007 J Neurochem. 2007 May; 101 (3): 749-56 and Wolfe et al. 2002, Neurology. 2002 May 14; 58 (9): 1435-6), optionally increased levels of autoantibodies to α-sinucrane in PD (Gruden et al. 2011, J Neuroimmunol. 2011 Apr; 233 (1-2): 221-7, Gruden et al. 2012 , Neuroimmunomodulation. 2012; 19 (6): 334-42 and Yanamandra 2011, PLoS One. 2011 Apr 25; 6 (4): e18513), or reduced self for α-sinucrane in PD patients compared to healthy controls. Antibodies were reported (Beson-Agbo et al 2013, Neurology. 2013 Jan 8; 80 (2): 169-75). It was suggested very soon after the discovery of this autoantibody that blood anti-α-synuclein autoantibodies could play a protective role in α-synuclein aggregation (Wolfe et al. 2002, Neurology. 2002 May 14). 58 (9): 1435-6).

Overexpression of α-synuclein in transgenic mice resembles some pathological aspects of Lewy body disease. Several different gene transfer systems for mice overexpressing α-synuclein have been generated in the last decade (described in the report below: Koehler et al 2014, PLoS One. 2013 May 31; 8 (5). ): E64649; Fleming and Chesselet, 2006, Behave Pharmacol. 2006 Sep; 17 (5-6): 383-91; Springer and Khale 2006, Curr Neuro Neurosci Rep. 6 (6); Mouse strains with They-1 and PDGF-β promoter develop motor and cognitive impairment and have been used to demonstrate the neuroprotective effects of antibodies against α-synuclein in vivo. However, there is no transgenetic system with robust degeneration of dopaminergic neurons, and in many cases the motor phenotype is driven by expression in motor neurons, which is usually not exacerbated in Parkinson's disease. Therefore, it is unclear whether the positive outcomes of potential disease-regulating therapies are mediated by effects on dopaminergic neurons or other central nervous system neurons.

One firm finding in the transgenic mouse model was that chronic overexpression of human α-synuclein impaired synaptic function. Studies in both in vitro and in vivo systems have shown that overexpression of wild-type (wt) human α-synuclein impaired synaptic transmission in the hippocampus (Nemani et al. 2010, Neuron. 2010 Jan 14). 65 (1): 66-79; Paumier et al. 2013, PLoS One. 2013 Aug 1; 8 (8): e70274). This was shown in the CA1 region of the hippocampus, where both studies found diminished basal synaptic transmission. The mechanism behind this was thought to be the intracellular accumulation of α-synuclein leading to synaptic release dysfunction. However, recent findings on the secretion of α-synuclein into the extracellular space at synapses and the toxic effects of α-synuclein oligomers on synaptic function open up the potential role of extracellular α-synuclein in synaptic dysfunction, which in turn opens up the potential role of extracellular α-synuclein in synaptic dysfunction. Therapeutic antibodies open the door to the ability to save disorders.

The use of viral vectors to overexpress α-synuclein is an important way to model rodent PD, because this method is still reproduced by gene mutations in mice or rats. This is because it causes a relatively rapid progressive degeneration of substantia nigra striatal neurons (Kirik and Bjorklund, 2003, Trends Neurosci. 2003 Jul; 26 (7): 386-92). In addition, viral gene delivery revealed the ability of wt α-synuclein to induce substantia nigra striatal lesions (Kirik et al. 2002, J Neurosci. 2002 Apr 1; 22 (7): 2780-91). Is a finding consistent with evidence of familial PD with α-synuclein duplication and triple duplication (Lee and Trojanowski, 2006, Neuron. 2006 Oct 5; 52 (1): 33-8). One study showed that a pool of goat antibodies against α-synuclein protected the N-terminus from dopaminergic cell death and ameliorated behavioral disorders in AAV-α-synuclein-based rat models of Parkinson's disease. (Shahaduzzaman et al 2015, PLoS One. 2015 Feb 6; 10 (2): e0116841).

Prion-like spread of α-synuclein lesions has recently been shown to express α-synuclein lesions and also dopaminergic cell death (Luk et al. 2012, Science. 2012 Nov 16; 338 (6109)). : 949-53). This model was used to show that α-synuclein antibody can ameliorate this lesion (Tran et al. 2014, Cell Rep. 2014 Jun 26; 7 (6): 2054-65). In this model, antibody treatment was able to reduce the accumulation of phosphorylated α-synuclein in some brain regions (including dopaminergic neurons in the substantia nigra) and reduce the development of motor disorders.

In addition to mutations, selective splicing of the α-synuclein gene and post-translational modification of proteins (such as phosphorylation, ubiquitination, nitration, and cleavage) enhance the aggregation and / or toxic form of α-synuclein. It can give rise to α-synuclein protein morphology with the ability (Beyer and Ariza, Mol Neurobiol. 2013 Apr; 47 (2): 509-24). However, the exact pathological species of α-synuclein remains unknown. Various misfolded / aggregated / secreted species, ranging from oligomers to fibrils, and various post-translational modifications have been associated with toxicity, but which is the most, even if there is actually one toxic species. There is no consensus on whether it is important.

Overall, the accumulation of α-synuclein with similar morphological and neurological changes in various animal models such as humans, mice, and flies makes this molecule central to the development of Lewy body disease. It suggests that it plays a role.

Several different antibodies to α-synuclein have been shown to have therapeutic effects in preclinical animal models. Both antibodies targeting epitopes containing α-synuclein residues 91-99 and antibodies targeting epitopes containing α-synuclein residues 118-126 confer effects on motor and cognitive disorders in transgenic mice. It has been shown that (Games et al. 2014, J Neuroscii. 2014 Jul 9; 34 (28): 9441-54). These state-of-the-art antibodies are humanized antibodies based on the mouse monoclonal antibody 9E4, which target an epitope containing α-synuclein residues 118-126 and are currently in Phase I clinical trials. C-terminal antibody 274 (Bae et al. 2012, J Neuroscii. 2012 Sep 26; 32 (39): 13454-69), which targets epitopes containing α-synuclein residues 120-140, is also a cell in the preclinical model. It has been shown to have an effect on the spread of lesions from to cells. In addition to these, antibodies targeting steric constellation species such as α-synuclein oligomers and fibrils have been shown to be able to at least reduce the levels of these presumably toxic α-synuclein species (Lindstroem). et al. 2014, Neurobiol Dis. 2014 Sep; 69: 134-43 and Speaker et al. 2014, Mol The 2014 Oct; 22 (10): 1753-67). These conformational antibodies, such as mab47, that reduce α-synuclein oligomer levels in vivo have also been shown to target epitopes at the C-terminus of α-synuclein from amino acids 121-125 (US patent application). Publication No. 20120308572). Other conformations, fibrils and oligomer-specific antibodies also target the C-terminal sequence (Vaikath et al. Neurobiol Dis. 2015; 79: 81-99).

Because the toxic form of α-synuclein is unknown, therapeutic antibodies are ideally the majority of α-synuclein species formed by alternative splicing or post-translational modifications (such as cleavage), as well as oligomers and fibrils. Should be able to bind to the morphology. As mentioned above, one problem with current antibodies being tested as therapeutic agents in preclinical models is that many of them have C-terminal epitopes that are not found in some of the major cleavage-type α-synucleins. That is what you are targeting. For example, the amino acids important for the binding of 9E4 are asparagine 122 and tyrosine 125 (according to the alanine scan shown in US Patent Application Publication No. 20140172713), which is the antibody in Parkinson's disease brain tissue. It means the inability to bind to α-synuclein cleaved at amino acids 119 and 122, which are part of the major cleavage species (Kellie et al. Sci Rep. 2014; 4: 5797). The same would be true for antibody 274 and antibody mab47 (US Pat. No. 8,632,776). Also, amino-terminal antibodies will probably not be able to bind to some of the major cleavage species lacking the first amino acid of α-synuclein, such as α-synuclein cleaved to amino acids 5-140. In the case of the 9E4 antibody, the mechanism of action was suggested to be the prevention of cleavage of amino acids 119-122 in the extracellular space, as the antibody binds to the same region where the protease cleaves α-synuclein (Gamees). et al. 2014, J Neurosci. 2014 Jul 9; 34 (28): 9441-54). A similar mechanism of action can be seen with antibodies in close proximity to this site, and therefore many antibodies around this region would be expected to have this activity.

There is some support for the toxic role of truncated α-synuclein species in animal models. Expression of cleaved α-synuclein under a tyrosine-hydroxylase promoter has been shown to lead to substantia nigra striatal lesions not normally seen in the transgenic α-synuclein model (Tofaris et al. 2006, J Neurosci. 2006 Apr 12; 26 (15): 3942-50; Wakamatsu et al. 2006, Neurobiol Aging. 2008 Apr; 29 (4): 574-85). For example, expression of amino acids 1-130 of the human α-synuclein protein with the A53T mutation caused embryo loss in dopaminergic neurons in the substantia nigra pars compacta, whereas expression of the full-length protein did not (). Wakamatsu et al. 2006, Neurobiol Aging. 2008 Apr; 29 (4): 574-85). Expression of the 120 amino acid α-synuclein molecule in a calcium / calmodulin-dependent protein kinase IIα (CamKII-α) promoter is associated with progressive impairment in cortical-hippocampal memory tests including α-synuclein aggregation and Burns labyrinth and novel object recognition. Associated (Hall et al. 2015, Exp Neurol. 2015 Feb; 264: 8-13). Also, in a rat AAV model, co-expression of C-terminally truncated α-synuclein promoted full-length α-synuclein-induced lesions (Ulusoy et al. 2010, Eur J Neurosci. 2010 Aug; 32 (3). ): 409-22).

In the present invention, antibodies (“GM37” and “GM285” described in Examples) capable of binding to toxic α-synuclein fragments 1-119 / 122 and abolishing this cleavage-type α-synuclein were produced. .. Antibodies of the invention, such as GM37 and GM285, are capable of binding to other oligomeric forms of α-synuclein and altering their uptake by other CNS resident cells to limit disease spread. .. Furthermore, the antibodies of the invention, such as GM37 and 285, were surprisingly found to be superior to prior art antibodies such as 9E4 in binding to different α-synuclein species in the human brain, and extracellular α -It has a surprisingly good effect on removing synuclein and normalizing synaptic transmission disorders induced by the presence of abnormal α-synuclein in vivo. To further explain their therapeutic potential, antibodies of the invention, such as GM37 and 285, can prevent the emergence of disease-related motor phenotypes in a rat model of Parkinson's disease. Finally, the antibodies GM37 and GM285 can inhibit seeding and phosphorylation of endogenous α-synuclein aggregation induced by extracellularly added recombinant pathological α-synuclein seeds in primary mouse neurons. .. Antibodies such as GM37 and 285 can also inhibit the seeding of α-synuclein lesions into dopaminergic neurons in vivo using a mouse model of Parkinson's disease, which prevents transmission of the lesion between cells. It further supports the therapeutic potential of these antibodies. Together, these data strongly support the use of these novel antibodies, GM37 and GM285, as new therapeutic agents capable of modifying the disease by inhibiting the mechanism by which disease lesions spread between neurons in Parkinson's disease patients. It is a thing.

In a further aspect of the invention, three amino acid variants of the GM37 antibody are provided. All variants have a functional readout similar to that of the parent antibody, GM37, but with improved manufacturability properties. These variants reduce the risk of post-translational modifications that occur within the binding region of the GM37 antibody and provide some improvement in antibody production. This is advantageous because large-scale clinical or commercial production of antibodies is complex and expensive, and providing a homogeneous product for the drug is particularly important for immunoglobulins and proteins.

The present invention provides novel monoclonal antibodies capable of specifically binding to an epitope within amino acids 112-117 (SEQ ID NO: 9 (ILEDMP)) in α-synuclein, and antigen-binding fragments thereof. Epitopes bound by an antibody of the invention or antibody binding fragments thereof, such as the exemplary antibody "GM37", or "GM285", are referred to herein as "112-117 epitopes". The antibodies of the invention specifically bind to epitopes within 112-117 epitopes and, according to one embodiment, may compete with antibodies GM37 or GM285 for binding to epitopes within amino acids 112-117. For example, the antibody or antigen-binding fragment thereof according to the present invention has a heavy chain consisting of the variable region of SEQ ID NO: 7 and a light chain consisting of the variable region of SEQ ID NO: 8, and is contained in amino acids 112 to 117 of human α-synuclein. It can compete for binding to the epitope. Inhibition of such competitive binding can be determined using assays and methods well known in the art, for example using unlabeled binding assays such as surface plasmon resonance (SPR). For example, human α-synuclein is immobilized on the surface and incubated with or without the reference antibody'GM37' prior to incubation with the antibody or binding fragment being tested. Alternatively, a pairwise mapping technique can be used, in which the reference antibody'GM37'is immobilized on the surface, the human α-synuclein antigen is bound to the fixed antibody, and then the secondary antibody is co-located to human α-synuclein. The binding ability is tested (see'BIAcore® Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05/2012, the disclosure of which is incorporated herein by reference).

More specifically, the GM285 antibody binds to an epitope within residues 112-117 of α-synuclein, including residues 112-115 of α-synuclein (ILED; SEQ ID NO: 19).

In one embodiment, the invention relates to the monoclonal antibody GM37, variants thereof (eg, GM37 variant 1, GM37 variant 2 and GM37 variant 3), or GM285.

In particular, the present invention provides a monoclonal antibody GM37, variants thereof (eg, GM37 variant 1, GM37 variant 2 and GM37 variant 3), or GM285, which can specifically bind to human synucleins. Such having a sufficient number (eg, 1, 2, or 3) of light chain CDRs and a sufficient number (eg, 1, 2, or 3) of heavy chain CDRs to form a binding site. Includes antibodies and derivatives thereof. Preferably, such antibodies have three light chain CDRs and three heavy chain CDRs, as defined below. Amino acid residues in this region may be numbered by IMGT®, International ImMunoGeneTechs Information System® or Kabat, E. et al. A. , Wu, T.I. T. , Perry, H. et al. M. , Gottesmann, K.K. S. & Foeller, C.I. (1991). Sexuals of Proteins of Immunological Interest, 5th edit. , NIH Publication no. 91-3242, US Department of Health and Human Services; Chothia, C.I. & Lesk, A. M. (1987). Canonical structures For the Hypervariable domains Of Immunogloblins. J. Mol. Biol. It is carried out according to 196, 901-917.

In one embodiment, the monoclonal antibody or antigen-binding fragment thereof can specifically bind to human α-sinuclein (a) heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 1; and / or (b) sequence. Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 2; and / or (c) Heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 3; and / or (d) Light chain CDR1 with the amino acid sequence of SEQ ID NO: 4; and / or (E) Light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and / or (f) Light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.
Has a synuclein antigen-binding fragment comprising or consisting of.

In another embodiment, the monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to human α-synuclein (a) heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1;
(B) Heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 33, 34 or 35;
(C) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3;
(D) Light chain CDR1 having the amino acid sequence of SEQ ID NO: 4;
(E) Light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and (f) Light chain CDR3 having the amino acid sequence of SEQ ID NO: 6;
Has a synuclein antigen-binding fragment comprising or consisting of.

In yet another embodiment, the monoclonal antibody or antigen-binding fragment thereof can specifically bind to human α-sinuclein (a) heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 20; and / or (b). ) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 21; and / or (c) Heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 22; and / or (d) Light chain CDR1 with the amino acid sequence of SEQ ID NO: 23; and / Or (e) light chain CDR2 having the amino acid sequence of SEQ ID NO: 24; and / or (f) light chain CDR3 having the amino acid sequence of SEQ ID NO: 25.
Has a synuclein antigen-binding fragment comprising or consisting of.

In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is different from that of the native anti-α-synuclein antibody and (compared to such a native anti-α-synuclein antibody):
(I) Difference in binding affinity (KD) for α-synuclein;
(Ii) Difference in ability of α-synuclein fibrils to inhibit protease cleavage;
(Iii) Differences in ability to ameliorate impaired basal synaptic transmission in F28-snca transgenic mice;
(Iv) Differences in ability to reduce levels of α-synuclein in mouse hippocampus as measured by in vivo microdialysis; and / or (v) Motor function in a rat model of Parkinson's disease when administered chronically Difference in ability to recover,
(Vi) Differences in ability to prevent seeding of α-synuclein (such as in vitro and / or accumulation of insoluble phosphorylated α-synuclein in a mouse model of Parkinson's disease); and / or (vi) cleavage in the human brain α -Has a synuclein antigen-binding fragment containing an amino acid sequence (in its CDR, its variable region, in its framework residues or in its constant region) indicating a difference in its ability to bind synuclein.

The antibodies of the invention and their antigen-binding fragments include Parkinson's disease (including (PD), idiopathic and hereditary Parkinson's disease), diffuse Lewy body disease (DLBD), and Lewy body variant Alzheimer's disease (LBV). , Can be used as a method for treating, diagnosing or imaging synucleinopathy such as Gaucher's disease (GD), complex Alzheimer's disease and Parkinson's disease (CAPD), pure autonomic failure and multiple system atrophy.

The immunization protocol for the production of hybridomas is shown. The table summarizes the differences in immunogens and mouse strains used to identify GM37 and GM285. Different HCo17-Balb / c and HCo12 / Balb / c mice were immunized separately (a description of these mice is shown below). Immunized with full-length α-synuclein containing amino acids 1-140 fibrils, cleaved α-synuclein fragments 1-60 and 1-119 of full-length (FL) α-synuclein (SEQ ID NO: 10) were administered (boosted). ) Hybridoma-expressing GM37 was identified from mice. The hybridoma expression antibody GM285 was an immunization protocol in which HCo12-Balb / c mice were immunized with the full-length monomer α-synuclein, amino acids 1-140, followed by the full-length fibrous α-synuclein (Example 1). ). (Panels A-C) show screening of GM37 for binding to α-synuclein, α-synuclein homologs and orthologs. A) Binding of antibody GM37 to α-synuclein using an unwashed solution-based ELISA (FMAT). B) Using SPR (Fortebio), binding of antibody GM37 is specific for α-synuclein (α-panel) and other related synuclein family proteins, β-synuclein (β-panel) and γ-synuclein (γ-panel). Does not join the panel). Measurements were made using SPR (Fortebio Octetred) and GM37 shows similar binding to α-synuclein from cynomolgus monkeys (cynomolgus monkey panels) and mice (mouse panels) (Example 1). C) Using SPR (Fortebio Octetredo), binding of antibody GM285 is specific for α-synuclein and does not bind to other related synuclein family proteins, β-synuclein and γ-synuclein. Measurements are made using SPR (Fortebio Octetred) to show similar binding of GM285 to α-synuclein from cynomolgus monkeys (cynomolgus monkeys) and mice (mice) (Example 1). (Panels A to C) show the real-time binding affinity of GM37. A) Binding of antibody GM37 to α-synuclein as measured by RU (Relative Unit) (y-axis) relative to time (X-axis) as determined by SPR (BIAcore® 3000). Goat anti-human IgG was immobilized on a CM5 chip. GM37 was captured on a goat anti-human IgG-immobilized chip and a series of concentrations of human α-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested for binding to the surface. did. The sensor surface was regenerated between each cycle. B) Signals from bonds at different concentrations that are converted into a bond curve. C) Calculated binding constant of antibody GM37 (denoted as hlgG1-6004-037-C106S) (Example 2). (Panels A to C) show the real-time binding affinity of GM285. A) Binding of antibody GM285 to α-synuclein as measured by RU (y-axis) with respect to time (X-axis) as determined by SPR (BIAcore® 3000). Goat anti-human IgG was immobilized on a CM5 chip. GM285 was captured on a goat anti-human IgG-immobilized chip and a series of concentrations of human α-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested for binding to the surface. did. The sensor surface was regenerated between each cycle. B) Signals from bonds at different concentrations that are converted into a bond curve. C) Calculated binding constant of antibody GM285 (denoted as hlgG1-6004-285) (Example 2). (Panels A to C) show real-time binding of comparative antibody 9E4. A) 9E4 binding to α-synuclein as measured by RU (y-axis) with respect to time (X-axis) as determined by SPR (BIAcore® 3000) is shown. Goat anti-human IgG was immobilized on a CM5 chip. 9E4 was captured on the chip by its binding to goat anti-human IgG immobilized on the chip. A series of concentrations of human α-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested for binding to the surface. The sensor surface was regenerated between each cycle. B) Signals from bonds at different concentrations that are converted into a bond curve. C) Calculated binding constants for antibody 9E4 (Example 2). The amino acid sequence of α-synuclein is shown. Major cleavage sites identified by mass spectrometry in human brain tissue (indicated by arrows) (Kellie JF, Higgs RE, Rider JW, Major A, Beach TG, Adler CH, Merchant K, Knierman MD. alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by mass spectrometery.SciRep.2014. (Panels A to B) show epitope mapping of antibodies GM37 and GM285. ELISA data showing the relative level of antibody binding to a contiguous peptide (20-mer) derived from the α-synuclein amino acid sequences 95-132 (other unbound peptides not shown). A) The GM37 epitope requires the peptide sequence ILEDMP (SEQ ID NO: 9) for complete binding. B) GM285 requires peptide ILED (SEQ ID NO: 19) for complete binding (Example 3). (Panels A to B) show a schematic diagram of a cut-type α-synuclein. A) The binding epitopes of GM37 / 285 (ILEDMP; SEQ ID NO: 9) and 9E4 (NEAYE; SEQ ID NO: 36) are shown in bold in the α-synuclein amino acid sequence (SEQ ID NO: 10). Arrows indicate the c-terminal cut site from FIG. B) The major truncated α-synuclein identified from human brain material. The size based on the number of amino acids is shown on the right. Full-length α-synuclein is 140 amino acids. As can be inferred from the epitope, GM37, its variants 1-3, and GM285 should bind to full length and 1-119 / 122, 1-135 fragments. Antibody 9E4 will bind only to full length and 1-135 fragments. The specificity of the smaller c-terminal fragment remaining after cleavage has not been shown. It is shown that the antibodies GM37 and GM285 immunoprecipitate full-length α-synuclein and truncated α-synuclein from the human brain. Crude homogenates of human DLB brains were subjected to test antibodies (beads (no antibody (No ab)), B12-human IgG1 control antibody that does not bind to α-synuclein, GM-37, GM37 variant 2, GM-285 and mice (m). ) 9E4) and immunoprecipitated material was isolated on SDS-PAGE after incubation with immunodepleted supernatant. Western blots show bands representing full-length and various truncated α-synucleins that have been depleted from the supernatant and immunoprecipitated with antibody (IP). As can be seen, the GM37, GM37v2 and GM285 antibodies deplete the major α-synuclein species from the supernatant, and the IP is these species, cleavage species 1-135, 1-119 / 122 and full-length α-synuclein. Is shown. 9E4 does not affect species 1-119 / 122, but only IP full length and 1-135 (Example 4). FIG. 6 shows a schematic diagram of proteolysis of α-synuclein fibrils cleaved by calpain at amino acids 119/122. Alpha-synuclein fibrils (PFF) are added to the culture containing (PFF +) or not containing the test antibody (PFF). The presence of GM-37 / 285 inhibits the formation of intracellular cleavage α-synuclein and its secretion into the cell medium. It is shown that GM37 inhibits the formation of cleavage bands (12KD) in the cytolysis of primary mouse cortical cultures treated with medium and PFF. Proteins were separated by SDS-PAGE and Western blotted to detect various species of α-synuclein. Two monomeric α-synuclein bands were detected at 12 and 14 kDa in cells treated with PFF alone or with control antibody (B12), representing cleavage and full-length α-synuclein, respectively. In the presence of GM-37, there is only a faint band at 12 Kd, indicating that most of the cleavage is inhibited. This effect is also reflected in the cell medium. The relative level of accumulation can also be inhibited by the presence of GM-37, as reflected in the decrease in relative intensity of the 14 Kd band. Alternatively, there may be a reduced amount of 14Kd band available for uptake by cells (Example 5). It shows dose-dependent inhibition of proteolysis of α-synuclein fibrils by antibodies GM37, GM37 mutant 2 and GM285. Bands representing full-length (FL) α-synuclein and bands representing C-terminal cleavage (CT) α-synuclein in cytolytic lysates from primary mouse cortical cultures at low antibody concentrations (0.1 ug / ml) (arrows) (Indicated by). Increasing the antibody concentration to 1, 5 and 10 ug / ml leads to a decrease in intracellular proteolysis of α-synuclein fibrils. This is observed with both antibodies GM37, GM37v2 and GM285. Control samples are treated with human IgG1 antibody B12, which does not recognize α-synuclein. There are also controls without antibody (without antibody) and cells without α-synuclein fibrils (without Synn). Also, the total amount of α-synuclein was reduced in samples treated with 37, 37v2 and 285 compared to B12 or "antibody-free" controls, which means that all three antibodies are concentration-dependently cellular. It is shown to reduce the accumulation of α-synuclein in. The actin band at the top of the gel indicates equal filling of the sample (Example 5). The effects of GM37 and GM285 on the seeding of α-synuclein aggregation and α-synuclein phosphorylation in mouse primary cortical neurons are shown. 12A) When cells are seeded with either 1 ng of pure or crude seeds of α-synuclein, the primary neurons stained for phosphorylated α-synuclein appear as intracellular spots or punctate stains. Image example. 12B) Western blot of protein from primary cortical neurons separated into soluble and insoluble fractions. Blots were taken with human α-synuclein-specific antibody (4B12 / Ha-sin), phospho-Ser-129-α-synuclein-specific antibody (ab51253 / pS-a-Syn) and mouse α-synuclein-specific antibody (D37A2). / M a-syn) staining and addition of crude seeds to primary neurons results in the accumulation of higher molecular weight multimers of endogenous mouse α-synuclein and phosphorylated α-synuclein and α-synuclein in the insoluble fraction. Show that it brings. 12C) GM37, GM37 variant 2 and GM285 inhibit the appearance of phosphorylated α-synuclein quantified as the number of intracellular α-synuclein phosphoserine 129 positive spots by Cellomics ARRAYSCAN ™ automatic microscopy. GM37, GM37v2 and GM285 dose-dependently reduce the amount of intracellular phosphorylated α-synuclein spots. 12D) Western blots of homogenates from primary cortical neurons treated with the highest dose of antibody (133 nM) and stained for actin, human α-synuclein, phosphorylated α-synuclein and mouse α-synuclein are antibodies 37, 37v2. And 285 show inhibition of cleavage of α-synuclein crude seeds taken up by cells in the insoluble fraction. Also, all antibodies inhibit phosphorylation in the insoluble fraction, accumulation of higher molecular weight multimers of endogenous mouse α-synuclein and phosphorylated mouse α-synuclein. The actin band at the top of the gel indicates equal filling of the sample (Example 6). It shows basal synaptic transmission at the Schaffer side branch-CA1 synapse in the hippocampus of F28-snca transgenic mice and age-matched control mice. Excitatory postsynaptic potential (fEPSP) was triggered by a single stimulus applied to the Schaffer side branch, and basal synaptic transmission was assessed by measuring the slope of fEPSP as a function of stimulus intensity. Short-term synaptic plasticity was assessed by induction of paired-pulse assessment. Stimulations of various intensities are 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 μA, which are added sequentially in ascending order and repeated 2-3 times at each intensity. Basal synaptic transmission was found to be significantly reduced in F28-snca transgenic mice overexpressing wild-type α-synuclein compared to age-matched control mice (Example 7). It shows the effect of a single dose of human 9E4 (15 mg / kg, intraperitoneal (ip)) systemically on impaired basal synaptic transmission at the Shaffer side branch-CA1 synapse in the hippocampus of F28-snca transgenic mice. Excitatory postsynaptic potential (fEPSP) was triggered by a single stimulus applied to the Schaffer side branch, and basal synaptic transmission was assessed by measuring the slope of fEPSP as a function of stimulus intensity. Acute treatment with h9E4 induced a significant improvement in impaired basal synaptic transmission in F28-snca transgenic mice (Tg-snca + h9E4 vs. Tg-snca + PBS, p = 0.002). However, the improvement with h9E4 was only partial as indicated by significantly lower basal synaptic transmission compared to PBS-treated littermates (p = 0.007) (Example 7). A single dose of human GM37 (15 mg / kg, intraperitoneal (ip)) or isotype-controlled antibody (B12) systemically for impaired basal synaptic transmission at the Shaffer side branch-CA1 synapse in the hippocampus of F28-snca transgenic mice. Shows the effect of administration. Excitatory postsynaptic potential (fEPSP) was triggered by a single stimulus applied to the Schaffer side branch, and basal synaptic transmission was assessed by measuring the slope of fEPSP as a function of stimulus intensity. Acute treatment with GM37 induced a complete amelioration of impaired basal synaptic transmission in F28-snca transgenic mice (Tg-snca + GM37 vs. Tg-snca + B12, p = 0.004) (Example 7). It shows the effect of a single dose of human GM285 (15 mg / kg, intraperitoneal (ip)) systemically on impaired basal synaptic transmission at the Shaffer side branch-CA1 synapse in the hippocampus of F28-snca transgenic mice. Excitatory postsynaptic potential (fEPSP) was triggered by a single stimulus applied to the Schaffer side branch, and basal synaptic transmission was assessed by measuring the slope of fEPSP as a function of stimulus intensity. Acute treatment with GM285 induced complete amelioration of impaired basal synaptic transmission in F28-snca transgenic mice (Tg-snca + GM285 vs. Tg-snca + PBS, p = 0.001) (Example 7). (Panels A-B) are systemic administrations of human 9E4, GM37 or isotype control antibody (anti-HEL) to levels of human α-synuclein in the interstitial fluid (isf) in the hippocampus of free-moving F28-snca transgenic mice. The effect (15 mg / kg, intraperitoneal (ip)) is shown. The average of 2 to 3 basal values (4 to 6 hours) prior to antibody treatment was taken as the baseline and set to 100% for each animal. Differences were analyzed using two-way ANOVA with repeated measures. The basal level of human α-synuclein in the hippocampus was 8.1 ± 1.1 ng / ml (mean ± SEM, n = 25, uncorrected for in vitro dialysis probe recovery). Administration of GM37 induced a greater reduction in human α-synuclein in the hippocampus of F28 mice compared to both the comparative antibody, human 9E4, and the control isotype, anti-HEL. Time points indicating significant differences in α-synuclein levels between animals or control antibodies treated with GM37 are indicated by the asterisks (Example 8). FIG. 19 shows a time-series schematic diagram of antibody treatment (down arrow), virus infusion and behavioral evaluation (Example 9) in the rat AAV human α-synuclein model shown in FIG. It is shown that antibody GM37 can reduce Parkinson's disease-like movement disorders after long-term treatment in a rat AAV model. The effect of long-term treatment with GM37 or PBS in AAV-human-α-synuclein rats on motor asymmetry will be assessed in a cylinder test. Each rat was tested for forefoot use by monitoring for 5 minutes. Percentages of right forefoot use (ipsilateral with infusion) and left forefoot use (opposite + right forefoot) were calculated for each animal compared to GFP-PBS rats (as shown on the y-axis) ^* , ^** p <0.05 and 0.01. Rats treated with PBS still have considerable asymmetry in paw use, while animals treated with antibody GM37 no longer have significant impairment (Example 9). It is shown that long-term treatment with antibody GM37 can reduce pathological α-synuclein phosphorylation induced by infusion of pathological α-synuclein fibrous seeds into the mouse striatum. FIG. 20A shows a schematic showing relative processing times for seed injection and cell counting. Antibody GM37 was administered 1 day prior to injection of recombinant α-synuclein fibrous seeds into the dorsal striatum of mice and then weekly for 6 weeks. The dosing regimen was either 15 mg / kg intravenous (iv) or 30 mg / kg intraperitoneal (ip). FIG. 20B shows the exposure level of GM37 in plasma based on the injection site and dose. Once a week, samples were taken prior to infusion of new antibody doses. FIG. 20C shows the exposure level (csf) of GM37 based on dose and injection site at the end of the study. FIG. 20D compares the number of cells to phosphorylated α-synuclein positive inclusion bodies counted in 6 portions in the substantia nigra after treatment with GM37 or PBS control. In mice treated with both 15 mg / kg intravenous (iv) and 30 mg / kg intraperitoneal (ip) GM37, phosphorylated α-synuclein inclusion bodies were intracellularly compared to mice treated with PBS. There was a considerable decrease in (Example 10). The alignment of human α (SEQ ID NO: 10), β (SEQ ID NO: 37) and γ (SEQ ID NO: 38) synuclein proteins is shown. Amino acid residues different from α-synuclein are highlighted. Gaps are indicated by dots. The SwissProt number is shown in parentheses. The alignment of α-synuclein ortholog (cynomolgus monkey, SEQ ID NO: 39; rat, SEQ ID NO: 40; mouse, SEQ ID NO: 41) is shown. Amino acid residues different from human α-synuclein (SEQ ID NO: 10) are highlighted. The SwissProt number is shown in parentheses. GM37 (named GM37 wild type (wt), and transient expression of three GM37 variants (named GM37 var 1, 2 and 3). Stars indicate protein A purification and medium data. It is shown that it is determined after summing. † indicates that the data are calculated from protein A and the yield obtained after neutralization, relative to the size of the expression culture (0.4 L). Shown is a competing ELISA measuring the binding of four antibodies to human α-synuclein, GM37 wt, GM37 var 1, GM37 var 2 and GM37 var 3. Using a plate coated with α-synuclein, the amount of antibody remaining after preincubation in a solution of each antibody (0.3 μg / ml) containing increasing concentrations of α-synuclein (0-1000 nM). To detect. All four antibodies show similar binding to α-synuclein. A table comparing the binding rate dynamic parameters of GM 37 wt and mutants 1-3 with immobilized recombinant human α-synuclein is shown. Binding was measured using SPR and binding rate was determined using a 1: 1 binding algorithm (BIAcore® T200). We compare the effects of α-synuclein antibodies on phosphorylated α-synuclein levels in primary mouse neurons treated with pathological α-synuclein fibrotic seeds. Primary neurons were treated with seeds (10 ng) in the presence or absence of four GM37, GM37 var 1, GM37 var 2 and GM37 var 3 (2 μg). Neurons were fixed and stained after 3 weeks and analyzed for α-synuclein phosphoserine 129 positive spots by Cellomics ARRAYSCAN ™. Cells treated with seed alone or with seed and isotype control antibody (B12) show significantly increased levels of phosphorylation. Cells treated with GM 37 wt and the three mutants can inhibit the phosphorylation of α-synuclein, all of which exhibit the same level of phosphorylation as unseeded cells. Data are shown as mean ± SD determined from 7 images per well in 5 wells. N = 2. The temperature-dependent aggregation of wt GM37, var1, var2 and var3 is compared. Each sample of antibody was steadily elevated in temperature over time and the level of aggregation was measured simultaneously by multiangle light scattering (Prometheus NT.48, NanoTemper Technologies). The temperature at which aggregation begins is similar for GM37 and GM37-mutants, but the lowest levels of aggregation are observed for GM37-Var2.

Definitions As used herein, the term "α-synuclein" is synonymous with "α-synuclein protein" and is identified in UniProt as an α-synuclein protein isoform (eg, P37840, 1-3). ). The amino acid numbering of α-synuclein is shown for SEQ ID NO: 10 as shown below, and methionine (M) is amino acid residue 1.

The present invention relates to α-synuclein, in particular antibodies and antibody fragments capable of specifically binding to human α-synuclein. In particular, antibodies and fragments thereof exhibit the ability to specifically bind to epitopes within 112-117 of human α-synuclein.

The term "antibody" (Ab) in the context of the invention is an immunoglobulin molecule capable of specifically binding to an epitope of a molecule ("antigen"), or, according to certain embodiments of the invention, an immunoglobulin. Refers to a fragment of a molecule. Native antibodies typically include a tetramer usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH in the present specification) and a heavy chain constant region composed of three regions (CH1, CH2 and CH3). Heavy chains can be of any isotype including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM and IgE. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain contains a κ chain and a λ chain. Heavy and light chain variable regions are typically involved in antigen recognition, while heavy and light chain constant regions are the first of various cells of the immune system (eg, effector cells) and classical complement systems. It can mediate the binding of immunoglobulins to host tissues or factors, including the component (C1q). The VH and VL regions can be further subdivided into hypervariable regions called "complementarity determining regions", which are interspersed with regions of more conserved sequences called "framework regions" (FRs). Each VH and VL consists of three CDR regions and four FR regions arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from amino-terminus to carboxy-terminus. The variable regions of the heavy and light chains contain binding regions that interact with the antigen. Of particular relevance are antibodies that are "isolated" to exist in a physical environment different from those that may exist in nature, or that are modified to differ from the natural antibodies in the amino acid sequence and those thereof. It is an antigen-binding fragment.

The term "epitope" means an antigenic determinant capable of specific binding to an antibody. Epitopes usually consist of surface groups of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural properties as well as specific charge properties. The conformational epitopes and linear epitopes are distinguished by the fact that their binding to the former, rather than the latter, is always lost in the presence of denaturing solvents. Epitopes are amino acid residues directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide (in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide). It may contain other amino acid residues that are not directly involved in binding. The term "112-117 epitope" refers to the region of human α-synuclein containing at least 4 of the 6 amino acid residues of 112-117 human α-synuclein, where 112-117 epitopes are human α-. Both residues 1-111 of synuclein (containing residues 118-111) and residues 118-140 of human α-synuclein (containing residues 118-120) Not included. As used herein, where an antibody is capable of specifically binding to human α-synuclein by binding to at least 4 of the 6 amino acid residues of the 112-117 epitope. , Antibodies are said to be capable of specifically binding to epitopes within "112-117 epitopes".

As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment, portion, region or domain of an antibody that can specifically bind to an epitope. It means whether it is produced as such (eg, by cleavage, by recombination, synthetically, etc.), and thus the term "antigen binding" means, for example, that "antigen binding fragment of antibody" is "antibody". It is intended to mean the same as "epitope binding", just as it is intended to be the same as "epitope binding fragment". Antigen binding fragments may contain all 1, 2, 3, 4, 5 or 6 of the CDR regions of such antibodies, although they are capable of specifically binding to such epitopes. It may exhibit specificity, affinity or selectivity for such epitopes that differ from those of such antibodies. However, preferably, the antigen binding fragment will contain all six of the CDR regions of such an antibody. The antigen-binding fragment of an antibody may be part of, or may include, a single polypeptide chain (eg, scFv), or an amino-terminal and carboxyl-terminal (eg, bispecific antibody, Fab fragment, etc.). It may be part of, or may contain, two or more polypeptide chains, each having a Fab ₂ fragment, etc.). Fragments of antibodies exhibiting antigen-binding ability are obtained, for example, by protease cleavage of intact antibodies. More preferably, two regions of the Fv fragment, VL and VH, are naturally encoded by separate genes, or polynucleotides encoding such gene sequences (eg, their coding cDNA) are VL and A single protein chain (known as a single chain Fv (scFv); for example, Bird et al., (1988) Science 242: 423-426; And Huston et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 5879-5883) as a flexible linker that allows them to be made. Can be combined using. Alternatively, bispecific antibodies by using flexible linkers (eg, less than about 9 residues) that are too short to allow the VL and VH regions of a single polypeptide chain to bind together. (Bispecific antibody), bispecific antibody (diabody), or similar molecule (two such polypeptide chains bind together to form a divalent antigen binding molecule) can be formed (this is a bivalent antigen binding molecule). See, for example, PNAS USA 90 (14), 6444-8 (1993) for a description of bispecific antibodies). Examples of antigen-binding fragments included within the scope of the invention are described in (i) Fab'or Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 regions, or WO 2007059782. Monovalent antibody; (ii) F (ab') 2 fragment, divalent fragment containing 2 Fab fragments linked by disulfide cross-linking in the hinge region; (iii) Fd fragment essentially consisting of VH and CH1 regions (Iv) Fv fragments essentially from the VL and VH regions, (v) essentially from the VH regions and also with domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov; 2i (ll): 484-90). The dAb fragments called dAb fragments (Ward et al., Nature 341, 544-546 (1989)); (vi) camelid or Nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5_ (l): l). ll-24) and (vii) isolated complementarity determining regions (CDRs). In addition, two regions of the Fv fragment, VL and VH, are encoded by separate genes, which are a single protein chain (single chain) in which the VL and VH regions are combined to form a monovalent molecule. As an antibody or single chain Fv (scFv), see, eg, Bird et al., Science 242,423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). It can be linked using recombinant methods by synthetic linkers that allow them to be made. These and other useful antibody fragments in the context of the present invention are further described herein. The term antibody is provided by antibody-like polypeptides such as chimeric and humanized antibodies, as well as any known technique such as enzymatic cleavage, peptide synthesis, and recombination techniques, unless otherwise specified. It should also be understood that it also includes antibody fragments that retain the ability to specifically bind to the fragment). The antibody produced can have any isotype. As used herein, "isotype" refers to an immunoglobulin class encoded by a heavy chain constant region gene (eg, IgG1, IgG2, IgG3 or IgG4). Such antibody fragments are obtained using conventional techniques known to those of skill in the art; suitable fragments capable of binding to the desired epitope are easily screened for usefulness as well as intact antibodies. obtain.

The term "bispecific antibody" refers to an antibody containing two independent antigen-binding fragments, each targeting an independent target. These targets can be epitopes present on different proteins, or different epitopes present on the same target. Bispecific antibody molecules can be made using compensatory amino acid modifications in the constant regions of HC of parent monospecific bivalent antibody molecules. The resulting heterodimer antibody contains one Fab given by two different parental monospecific antibodies. Amino acid modifications in the Fc region result in improved stability of heterodimeric antibodies with stable bispecificity over time (Ridgway et al., Protein Enginering 9, 617-621 (1996), Gunasekaran et. al., JBC 285,1967-1 (2010), Moore et al., MAbs 3: 6 546-557 (2011), Strop et al., JMB 420, 204-219 (2012), Metz et al., Protein Engineing 25:10 571-580 (2012), Labridn et al., PNAS 110: 113, 5145-5150 (2013), Splitter Von Protein et al., MAbs 5: 5 646-654 (2013). Bispecific antibodies can also include molecules produced using ScFv fusion. The two unispecific scfvs are then independently bound to an Fc region capable of forming a stable heterodimer to produce a single bispecific molecule (Mabry). et al., PEDS 23: 3 115-127 (2010). Bispecific molecules have dual binding capacity, eg, deliver therapeutic antibodies across the blood-brain barrier to treat CNS disorders. To target both therapeutic targets and transcellular transport surface receptors.

The terms GM37, GM-37, GM37 wild type (wt), mab37 and 6004-37 are used interchangeably herein to refer to the same antibody.

The term antibody GM37 is intended to include antibodies or antigen-binding fragments thereof comprising or consisting of the heavy chains set forth in CDR1-3 SEQ ID NOs: 1-3 and the light chains CDR1-3 set forth in SEQ ID NOs: 4-6. To. In one embodiment, antibody GM37 or an antigen binding fragment thereof may include or consist of a heavy chain variable region of SEQ ID NO: 7 and / or a light chain variable region of SEQ ID NO: 8. For example, antibody GM37 is an IgG antibody comprising a light chain consisting of the variable region of SEQ ID NO: 8 and the κ constant region of SEQ ID NO: 17 as well as a heavy chain consisting of the variable region of SEQ ID NO: 7 and the constant region of SEQ ID NO: 18. possible.

Deamination of proteins, in these cases antibodies, can occur spontaneously during production and storage, as well as in vivo, making it difficult to control the quality of the final drug. Deamination can also affect the activity of the molecule in some cases. Deamination occurs at asparagine residues, but the location of the associated asparagine can be difficult to predict with certainty, but in some cases can be influenced by the asparagine-glycine motif. Although several possible deamination motifs are found on the GM37 antibody, one site of potential deamination was found to be heavy chain residue 54. Subsequent substitution of asparagine with another amino acid is not trivial and the three variants of GM37 (GM37 variants (var) 1, 2 and 3) have the activity of the original GM37 (GM37 wild type (wt)). Turned out to hold.

The term GM37 variant refers to deaminated mutants 1, 2 or 3, where variant 1 has an N54S substitution as compared to the GM37 antibody described above herein. Mutant 2 has N54Q substitution and variant 3 has N54H.

Here, the antibodies GM37 variants (var) 1, 2 and 3 are the heavy chains set forth in CDR1 and 3 SEQ ID NOs: 1 and 3 from GM37 and the light chain CDR1-3 from GM37 set forth in SEQ ID NOs: 4-6. Such that variant 1 has CDR2 of SEQ ID NO: 33, variant 2 has CDR2 of SEQ ID NO: 34, and variant 3 has CDR2 of SEQ ID NO: 35. , Their heavy chain CDR2 is intended to contain an antibody or an antigen-binding fragment thereof.

In one embodiment, the antibody GM37 variant or its antigen binding fragment comprises the heavy chain variable regions of SEQ ID NOs: 30, 31 and 32, respectively, and the light chain variable regions of SEQ ID NO: 8 for variants 1, 2 and 3, respectively. Can include or consist of it. The antibody GM37 contains an IgG comprising a light chain consisting of the variable region of SEQ ID NO: 8 and the κ constant region of SEQ ID NO: 17, as well as a heavy chain consisting of the variable region of SEQ ID NO: 30, 31 or 32 and the constant region of SEQ ID NO: 18. It can be an antibody.

The terms GM285, GM-285, mab285 and 6004-285 are used interchangeably herein to refer to the same antibody.

The term antibody GM285 is intended to include an antibody or antigen-binding fragment thereof comprising or consisting of heavy chains set forth in CDR1-3 SEQ ID NOs: 20-22 and light chains CDR1-3 set forth in SEQ ID NOs: 23-25. Will be done. In one embodiment, antibody GM37 or an antigen binding fragment thereof may comprise or consist of a heavy chain variable region of SEQ ID NO: 26 and / or a light chain variable region of SEQ ID NO: 27. For example, antibody GM37 is an IgG antibody comprising a light chain consisting of the variable region of SEQ ID NO: 27 and the κ constant region of SEQ ID NO: 29, as well as a heavy chain consisting of the variable region of SEQ ID NO: 26 and the constant region of SEQ ID NO: 28. possible.

The GM285 antibody specifically binds to an epitope within sequences 112-115 (ILED; SEQ ID NO: 19) of human α-synuclein (SEQ ID NO: 10).

Unless otherwise specified herein, amino acid residue numbering in this region may be described by IMGT®, International ImMunoGeneTechs Information System® or Kabat, E. et al. A. , Wu, T.I. T. , Perry, H. et al. M. , Gottesmann, K.K. S. & Foeller, C.I. (1991). Sexuals of Proteins of Immunological Interest, 5th edit. , NIH Publication no. 91-3242, US Department of Health and Human Services. Chothia, C.I. & Lesk, A. M. (1987). Canonical structures for the hypervariable domains of immunogloblins. J. Mol. Biol. It is carried out according to 196, 901-917).

"Anti-α-synuclein antibody" or "α-synuclein antibody" (used interchangeably herein, as used herein) is α-synuclein or α as defined above herein. -Synuclein fragment, in particular an antibody or antigen-binding fragment thereof that specifically binds to the sequence of α-synuclein corresponding to SEQ ID NOs: 9 and / or 19.

As used herein, the term "human antibody" (which may be abbreviated as "humAb" or "HuMab") includes antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Is intended. Human antibodies of the invention may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, by random or site-specific mutations in vitro, or during gene rearrangements, or somatic mutations. Mutations induced by).

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to the preparation of antibody molecules of a single molecular composition. Traditional monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. In certain embodiments, the monoclonal antibody can be composed of more than one Fab region, thereby increasing its specificity for more than one target. The term "monoclonal antibody" or "monoclonal antibody composition" is not intended to be limited by any particular production method (eg, recombination, transgenic, hybridoma, etc.).

The term "humanization" refers to antigen binding sites obtained from non-human species-derived immunoglobulins, commonly prepared using recombinant techniques, and the remaining immunoglobulins based on the structure and / or sequence of human immunoglobulins. Refers to a molecule with a structure. The antigen binding site is only the complete non-human antibody variable region fused to the human constant region, or the complementarity determining regions (CDRs) of such variable regions transplanted into the appropriate human framework regions of the human variable region. Can include either. Framework residues of such humanized molecules may be wild-type (eg, fully human), or they are one that is not found in human antibodies whose sequences functioned as the basis for humanization. Alternatively, it can be modified to include multiple amino acid substitutions. Humanization reduces or eliminates the likelihood that the constant region of the molecule acts as an immunogen in a human individual, but the potential for an immune response to the foreign variable region remains (LoBuglio, AF et al. (1989) “Mouse / Human Molecular Molecular Antibody In Man: Kinetics And Immune Response”, Proc. Natl. Acad. Sci. (USA) 86: 4220-4224). Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable regions to reshape them as close as possible to the human form. Both heavy and light chain variable regions contain three complementarity determining regions (CDRs) that differ in their response to the antigen of interest and determine their ability to bind, and these complementarity determining regions (CDRs) are located. It is known that four framework regions (FRs) that are relatively conserved in a given species and are thought to provide a scaffolding for CDRs are adjacent. When a non-human antibody is prepared for a particular antigen, the variable region is "reformed" or by transplanting the CDR derived from the non-human antibody into the FR present in the modified human antibody. Can be "humanized". The application of this technique to various antibodies is described by Sato, K. et al. et al. (1993) Cancer Res 53: 851-856. Richmann, L. et al. et al. (1988) "Reshaping Human Antibodies for Therapy", Nature 332: 323-327; Verhoeyen, M. et al. et al. (1988) “Reshapping Human Antibodies: Grafting An Antibodies Activity”, Science 239: 1534-1536; Kettlebough, C.I. A. et al. (1991) "Humanization Of A Mouse Monoclonal Antibodies By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation", Protein733-E. et al. (1991) “Construction Of Reshaved Human Antibodies With HIV-Neutralizing Activity”, Human Antibodies Hybridoma 2: 124-134; Gorman, S. et al. D. et al. (1991) “Reshaping A Therapeutic CD4 Antibodies”, Proc. Natl. Acad. Sci. (USA) 88: 4181-4185; Tempest, P.M. R. et al. (1991) “Respirating A Human Monoclonal Antibodies To Inhibit Human Respiratory Syncytial Virus In vivo”, Bio / Technology 9: 26. S. et al. (1991) “Humanized Antibodies For Antiviral Therapy”, Proc. Natl. Acad. Sci. (USA) 88: 2869-2873; Carter, P. et al. et al. (1992) "Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy", Proc. Natl. Acad. Sci. (USA) 89: 4285-4289; and Co, M. et al. S. et al. (1992) "Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen", J. Mol. Immunol. It is reported by 148: 1149-1154. In certain embodiments, the humanized antibody preserves all CDR sequences, eg, a humanized mouse antibody comprising all six CDRs derived from the mouse antibody. In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) modified relative to the original antibody, which are these. Is also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. The ability to humanize an antigen is well known (eg, US Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5. , 859, 205; see 6,407,213; see 6,881,557).

As used herein, an antibody or antigen-binding fragment thereof is associated with that epitope more frequently, more rapidly, for a longer period of time and / or with higher affinity or binding activity compared to another epitope. When reacting or associating, it is said to "specifically" bind to the region of another molecule (ie, an epitope). In one embodiment, the antibody of the invention, or antigen-binding fragment thereof, binds at least 10-fold stronger to its target (human alpha-synuclein) than another molecule; preferably at least 50-fold stronger, more preferably at least 100-fold stronger. do. Preferably, the antibody, or antigen-binding fragment thereof, binds under physiological conditions, eg, in vivo. Thus, an antibody capable of "specifically binding" to an epitope within residues 112-117 (ILEDMP (SEQ ID NO: 9)) of human α-synuclein has such specificity and / or such. Includes an antibody or antigen-binding fragment thereof capable of binding to an epitope within residues 112-117 of human α-synuclein under conditions. Suitable methods for determining such binding are known to those of skill in the art, and exemplary methods are described in the accompanying examples. As used herein, the term "binding" in the context of binding an antibody to a given antigen typically refers to BIAcore® 3000 or T200 using the antigen as a ligand and the antibody as a sample. Binding with affinity corresponding to KD of about ^10-7 M or less, such as about ^10-8 M or less, such as about ^10-9 M or less, as determined by, for example, surface plasmon resonance (SPR) technology in any of the instruments. Refers to, at least 10-fold lower, eg, at least 100-fold lower, eg at least 1, It binds to a given antigen with an affinity corresponding to KD, which is 000-fold lower, eg, at least 10,000-fold lower, eg, at least 100,000-fold lower. The amount of lower affinity depends on the KD of the antibody, so if the KD of the antibody is very low (ie, the antibody is very specific), the affinity for the antigen will be the affinity for the non-specific antigen. The amount that is lower than sex can be at least 10,000 times.

As used herein, the term "kd" (seconds-1 or 1 / second) refers to the dissociation rate constant of a particular antibody-antigen interaction. The value is also called a koff value.

As used herein, the term "ka" (M-1 x s-1 or 1 / M s) refers to the binding rate constant of a particular antibody-antigen interaction.

As used herein, the term "KD" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing kd by ka.

As used herein, the term "KA" (M-1 or 1 / M) refers to the binding equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing ka by kd.

In one embodiment, the invention has the following characteristics:
i. Binding affinity (KD) for 0.5-10 nM α-synuclein, such as 1-5 nM or 1-2 nM;
ii. Ability to inhibit protease cleavage of α-synuclein fibrils;
iii. Ability to ameliorate impaired basal synaptic transmission in F28-snca transgenic mice;
iv. Ability to reduce levels of α-synuclein in mouse hippocampus as measured by in vivo microdialysis;
v. Ability to restore motor function in a rat model of Parkinson's disease when administered chronically vi. Ability to prevent seeding of α-synuclein, such as in vitro and / or accumulation of insoluble phosphorylated α-synuclein in a mouse model of Parkinson's disease; and / or vii. With respect to an antibody or antigen-binding fragment thereof that exhibits one or more of the ability to bind to cleavage α-synuclein in the human brain.

The binding affinity (KD) for α-synuclein can be determined, for example, using a method well known in the art described in Example 2.

The term "ability to inhibit protease cleavage of α-synuclein fibrils" includes the ability to inhibit calpain-1 -induced formation of human α-synuclein fragments 1-119-122 in primary cortical neurons (Example 5). See).

The term "ability to ameliorate impaired basal synaptic transmission in F28-snca transgenic mice" is used in F28-snca transgenic mice, as indicated, for example, by the tilt of fEPSP induced when measured electrophysiologically. Includes the ability to improve synaptic transmission and reduced plasticity in the CA1 region of the hippocampus (see Example 6).

The term "ability to reduce levels of α-synuclein in the mouse hippocampus as measured by in vivo microdialysis" refers to human α in hippocampal arousal, free-moving F28-snca transgenic mice when measured using in vivo microdialysis. Includes the ability to reduce levels of synuclein (see Example 7).

The term "ability to restore motor function in a rat model of Parkinson's disease when administered chronically" reduces or eliminates motor asymmetry in a rat model of Parkinson's disease recombinant adeno-associated virus vector (rAAV). Includes capabilities (see Example 8).

In some antibodies, a small portion of the CDR, a subset of the CDR residues required for binding, called SDR, is required to retain binding in the humanized antibody. CDR residues that do not contact the relevant epitope and are not in the SDR are outside the Chothia hypervariable loop, based on previous studies (eg, residues H60-H65 in CDR H2 are often not required). From the area of Kabat CDR in (Kabat et al. (1992) SEQENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Research Network No. 91-3242; Of Immunoglobulins ”, J. Mol. Biol. 196: 901-917), by molecular modeling and / or by experiment, or by Gonzales, N. et al. R. et al. (2004) "SDR Gruffing Of A Murine Antibody Using Multiple Human Germline Temples To Minimize Its Immunogenicity", Mol. Immunol. It can be specified as described in 41: 863-872. In such humanized antibodies at positions where one or more donor CDR residues are absent or where all donor CDRs are omitted, the amino acids occupying this position correspond (by Kabat numbering) in the receptor antibody sequence. Can be an amino acid that occupies a position. The number of such substitutions of the receptor for the donor amino acid in the included CDR reflects a balance of competing considerations. Such substitutions are potentially advantageous in reducing the number of mouse amino acids in humanized antibodies and, as a result, reducing their potential immunogenicity. However, substitutions can also cause changes in affinity, and it is preferable to avoid a significant decrease in affinity. The position of the substitution in the CDR and the amino acid to be substituted can also be selected experimentally.

A single amino acid modification of a CDR residue can result in loss of functional binding (Radikoff, S. etc. (1982) "Single Amino Acid Substition Altering Antigen-Binding Specificity", Proc. Natl. (USA) 79 (6): 1979-1983) provide a means for systematically identifying another functional CDR sequence. In one preferred method for obtaining such mutant CDRs, the polynucleotide encoding the CDR is mutated (eg, by random mutation or by a site-specific method (eg, encoding a mutation locus). By polymerase chain-mediated amplification)), CDRs with substituted amino acid residues are produced. By comparing the identity of the relevant residues in the original (functional) CDR sequence with the identity of the substituted (non-functional) mutant CDR sequence, BLOSUM62. The iij substitution score can be identified. The BLOSUM system provides a matrix of amino acid substitutions created by analyzing a database of sequences for reliable alignment (Eddy, S.R. (2004) "Here Did The BLOSUM62 Alignnment Score Matrix Come From?", Nature. Biotech. 22 (8): 1035-1036; Henikoff, J.G. (1992) "Amino acid substation matrix from blocks", Proc. Natl. Acad. Sci. (USA) 89: 10915-10919K. . Et al. (1990) "Methods For Assessing The Static Scientific Of Molecular Sequence Features By Using General Scoring Schemes" (Alc26 ) "Amino Acid Substation Matrix From Information Theoretic Perspective", J. Mol. Biol. 219, 555-565. Currently, the state-of-the-art BLOSUM database is BLOSUM62 database (BLOSUM62). Shows an iij substitution score (the higher the score, the more conservative the substitution, and thus the more likely that the substitution will not affect function). The resulting CDR-containing antigen-binding fragment binds to α-sinucrane. If not, for example, the BLOSUM62.iii substitution score is considered to be inadequately conservative and new substitution candidates with higher substitution scores are selected and generated, thus, for example, the original residue. If it is glutamate (E) and the non-functional substitution residue is histidine (H), the BLOSUM62.iii substitution score is 0 and more conservative changes (aspartate, asparagine, glutamine, or lysine, etc.) To) is preferable.

Therefore, the present invention envisions the use of random mutations to identify improved CDRs. In the context of the present invention, conservative substitutions can be defined by substitutions within the range of amino acid types reflected in one or more of the three tables below.

More conservative substituents include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

Further groups of amino acids are described, for example, in Creighton (1984) Proteins: Structure and Molecular Properties (2d Ed. 1993), W. et al. H. It can be formulated using the principles described in Freeman and Company.

Phage display techniques can be used instead to increase (or decrease) CDR affinity. This technique, called affinity maturation, uses mutations or "CDR walking" and re-selection using the target antigen or its antigenic antigen-binding fragment to the initial antibody or parent. Identify antibodies with a CDR that binds with a higher (or lower) affinity for the antigen when compared to the antibody (see, eg, Glasser et al. (1992) J. Immunology 149: 3903). Mutagenesis of the entire codon rather than a single nucleotide results in a semi-randomized repertoire of amino acid mutations. A library consisting of a pool of mutant clones can be constructed, each of which is dependent on a single amino acid modification in a single CDR and presents each possible amino acid substitution for each CDR residue. Includes mutants. Mutants with increased (or decreased) binding affinity for the antigen can be screened by contacting the immobilized mutant with the labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased or decreased affinity for the antigen (eg, ELISA) (Wu et al. 1998, Proc. Natl). .Acad.Sci. (USA) 95:6037; see Yelton et al., 1995, J. Immunology 155: 1994). CDR walking that randomizes the light chain may be available (see Schier et al., 1996, J. Mol. Bio. 263: 551).

Methods for achieving such affinity maturation include, for example: Krause, J. et al. C. et al. (2011) “An Insertion Mutation That Distorts Antibodies Binding Site Architecture Elements Function Of A Human Antibodies”, MBio. 2 (1) pii: e00345-10. doi: 10.1128 / mBio. 00345-10; Kuan, C.I. T. et al. (2010) “Affinity-Matured Anti-Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas And Melanomas”, Int. J. Cancer 10.1002 / ijc. 25645; Hackel, B. et al. J. et al. (2010) “Stability And CDR Composition Biases Engineering Binder Functionality Landscapes”, J. Mol. Mol. Biol. 401 (1): 84-96; Montgomery, D.I. L. et al. (2009) “Affinity Maturation and characterization Of A Human Monoclonal Antibody Against HIV-1 gp41”, MAbs 1 (5): 462-474; Gustchina, E. et al. et al. (2009) "Affinity Maturation By Targeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab Derived From A Synthetic Naive Human Antibody Library And Directed Against The Internal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With Improved HIV-1 Neutralization Potency And Breadth ”, Virology 393 (1): 112-119; Finally, W. J. et al. (2009) "Affinity Mutagenesis Of A Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Leveter Engine Engine Mol. Biol. 388 (3): 541-558; Bostrom, J. Mol. et al. (2009) “Improving Antibody Binding Affinity And Specificity For Therapeutic Development”, Methods Mol. Biol. 525: 353-376; Steidl, S. et al. et al. (2008) "In Vitro Affinity Measurement Of Human GM-CSF Antibodies By Targeted CDR-Diversification", Mol. Immunol. 46 (1): 135-144; and Barderas, R. et al. et al. (2008) “Affinity Maturation Of Antibodies Assisted By In silico Modeling”, Proc. Natl. Acad. Sci. (USA) 105 (26): 9029-9034.

Thus, the sequences of the CDR variants of the included antibodies or their antigen binding fragments are by substitution; for example, 4 amino acid residues, 3 amino acid residues, 2 amino acid residues or 1 amino acid residue to be substituted. Can differ from the sequence of the CDR of the parent antibody, GM37, GM37 var 1-3, or 285. According to one embodiment of the invention, it is further envisioned that the amino acids in the CDR regions can be replaced by conservative substitutions as defined in the three tables above.

As used herein, the term "treatment" or "treating" is used to improve, delay, alleviate, or suppress the progression or severity of a disease or disorder. Or it means ameliorating, delaying, alleviating, or suppressing one or more symptoms or side effects of such a disease or disorder. In the spirit of the present invention, "treatment" or "treating" further means a method for obtaining a beneficial or desired clinical result, wherein the "beneficial or desired clinical result" is limited. Not done, but partial or global, detectable or undetectable, alleviating symptoms, reducing the degree of disability or illness, stable (ie, not exacerbated) These include delaying or slowing the progression of the disease or disorder, amelioration or alleviation of the disease or disorder, and remission of the disease or disorder.

An "effective amount", when applied to an antibody of the invention or an antigen-binding fragment thereof, is required to achieve the intended biological effect or desired therapeutic outcome (including, but not limited to, clinical outcome). Refers to a sufficient amount over a reasonable dose and duration. The phrase "therapeutically effective amount", when applied to an antibody of the invention or an antigen-binding fragment thereof, improves or alleviates the progression of a disorder or disease state, or the progression of a disorder or disease symptom. It is intended to represent the amount of antibody, or antigen-binding fragment thereof, sufficient to stabilize, suppress, delay, alleviate, or delay. In one embodiment, the method of the invention provides administration of an antibody, or antigen-binding fragment thereof, in combination with another compound. In such cases, the "effective amount" is the amount of combination sufficient to cause the intended biological effect.

Therapeutically effective amounts of the anti-α-synuclein antibody or antigen-binding fragment thereof of the present invention include the condition, age, sex, and body weight of the individual, and the ability of the anti-α-synuclein antibody to elicit the desired response in the individual. It can change depending on the factors. A therapeutically effective amount is also an amount that outweighs any toxic or adverse effects of the antibody or antibody portion by a therapeutically beneficial effect.

As shown above, the invention relates specifically to monoclonal antibodies capable of specifically binding to an epitope within amino acids 112-117 (SEQ ID NO: 9 (ILEDMP)) of human α-synuclein. In one embodiment, the antibody is capable of competing with antibody GM37 for binding of α-synuclein to an epitope within the 112-117 amino acids.

The antibodies of the invention exemplified by GM37, its variants GM37 var 1-3 and GM285, and their α-synuclein binding fragments are toxic α-synuclein fragments consisting of residues 1-119 / 122 of α-synuclein. It is possible to bind and neutralize its toxicity (eg, by binding extracellularly to the α-synuclein fragment, thereby preventing it from being taken up by the cell. Surprisingly, α-synuclein. The antibody of the present invention capable of binding to an epitope in amino acids 112 to 117 of the above is superior to prior art antibodies such as antibody 9E4 in binding to toxic α-synuclein species in the human brain, and extracellular α. -It has an excellent effect on removing synuclein and normalizing α-synuclein-induced synaptic transmission disorders in vivo. The antibodies of the present invention show the appearance of related motor phenotypes in a rat model of Parkinson's disease. It can also be improved.

The antibody of the present invention is preferably a human or humanized antibody.

The present invention is a method of reducing α-synuclein aggregate formation in a patient, and also includes a method of administering a therapeutically effective amount of the antibody of the present invention to a patient in need of such treatment. offer.

In addition, the antibody can be in the composition with a pharmaceutically acceptable carrier, diluent and / or stabilizer. The antibodies of the invention can be used therapeutically. In particular, the antibodies of the invention include Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gaucher's disease, diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBV), and combined Alzheimer's disease. It can be used to treat diseases and synucleinopathy such as Parkinson's disease, pure autonomic failure and multiple system atrophy.

The treatment envisioned by the present invention may be long term and the patient may be treated for at least 2 weeks, eg at least 1 month, 6 months, 1 year or more.

Antibodies to the antigen-binding fragments of the invention can be obtained from various cell lines such as human cell lines, non-human mammalian cell lines, and insect cell lines such as CHO cell lines, HEK cell lines, BHK-21 cell lines, mouse cells. Strains (myeloma cell lines, etc.), fibrosarcoma cell lines, PER. It can be produced in C6 cell lines, HKB-11 cell lines, CAP cell lines and HuH-7 human cell lines (contents are incorporated herein by reference, Dumont et al, 2015, Crit Rev Biotechnol. Sep 18: 1-13.).

The antibody of the present invention is, for example, Kohler et al. , Nature 256,495 (1975) may be a monoclonal antibody produced by the hybridoma method first described, or may be produced by a recombinant DNA method. Monoclonal antibodies are also described, for example, in Clackson et al. , Nature 352,624-628 (1991) and Marks et al. , J. MoI. Biol. It can be isolated from a phage antibody library using the technique described in 222,581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies are prepared from mouse spleen B lymphocytes obtained from mice immunized with the antigen of interest, for example, in the form of cells expressing the antigen on the surface or nucleic acids encoding the antigen of interest. Obtained from the hybridoma. Monoclonal antibodies are also obtained from hybridomas derived from antibody-expressing cells of immunized human or non-human mammals (rats, rabbits, dogs, sheep, goats, primates, etc.).

In one embodiment, the antibody of the invention is a human antibody. Human monoclonal antibodies to α-synuclein can be produced using transgenic or chromosome-introduced mice that have a portion of the human immune system rather than the mouse system. Such transgenic and chromosome-introduced mice include mice referred to herein as HuMAb mice and KM mice, respectively.

HuMAb mice have unrearranged human heavy and constant (μ and Y) and light chain variable and constant (κ) chains, with targeted mutations that inactivate the endogenous μ and κ chain loci. It contains a minilocus of a human immunoglobulin gene encoding an immunoglobulin sequence (Lomberg, Net al., Nature 368, 856-859 (1994)). Thus, mice exhibit reduced expression of mouse IgM or Igκ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutations, resulting in high affinity. Human IgG, κ monoclonal antibody produced (Lomberg, Net al. (1994), see above; Lomberg, N., Handbook of Experimental Pharmaceutical 113, 49-101 (1994), Lomberg, N. and Huszar, D. , Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lomberg, N., Ann. NY. Acad. Sci 764 536-546 (1995)). .. HuMAb mice were prepared by Taylor, L. et al. et al. , Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. Mol. et al. , International Immunology 5,647-656 (1993), Tuaillon et al. , J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al. et al. , International Immunology 6,579-591 (1994), Fishwild, D. et al. et al. , Nature Biotechnology 14,845-851 (1996). US Pat. No. 5,545,806, US Pat. No. 5,569,825, US Pat. No. 5,625,126, US Pat. No. 5,633,425, US Pat. 5,789,650, US Pat. No. 5,877,397, US Pat. No. 5,661,016, US Pat. No. 5,814,318, US Pat. No. 5, , 874,299, US Pat. No. 5,770,429, US Pat. No. 5,545,807, International Publication No. 98/24884, International Publication No. 94/25585, See also International Publication No. 93/1227, International Publication No. 92/22645, International Publication No. 92/03918 and International Publication No. 01/09187.

HCo7, HCo12, HCo17 and HCo20 mice have JKD disruption in their endogenous light chain (κ) gene (as described in Chen et al., EMBO J.12, 811-820 (1993)), they. CMD disruption in the endogenous heavy chain gene (as described in Example 1 of WO 01/14424), and the KCo5 human kappa light chain transgene (Fishwild et al., Nature Biotechnology 14,845). -As described in 851 (1996)). In addition, HCo7 mice have the HCo7 human heavy chain transgene (as described in US Pat. No. 5,770,429), and HCo12 mice have the HCo12 human heavy chain transgene (International Publication No. 5). The HCo17 mouse has the HCo17 human heavy chain transgene (as described in Example 2 of the 01/14424 pamphlet) as described in Example 2 of the International Publication No. 01/09187 pamphlet. The HCo20 mouse has the HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy chain and κ light chain transgenes in background homozygotes due to disruption of the endogenous mouse heavy chain and κ light chain loci.

In the KM mouse strain, the endogenous mouse kappa light chain gene is described in Chen et al. , EMBO J. Homozygous disruption as described in 12,811-820 (1993), the endogenous mouse heavy chain gene is as described in Example 1 of WO 01/09187. It is homozygously destroyed. This mouse strain was described in Fishwild et al. , Nature Biotechnology 14,845-851 (1996), which carries the human kappa light chain transgene, KCo5. This mouse strain also carries a human heavy chain introduction chromosome composed of chromosome 14 fragment hCF (SC20), as described in WO 02/4348. HCo12-Balb / c, HCo17-Balb / c and HCo20-Balb / c mice were described as HCo12, HCo17 and HCo20 in KCo5 [J / K] (as described in International Publication No. 09/097006. It can be generated by crossing Balb).

In the KM mouse strain, the endogenous mouse kappa light chain gene is described in Chen et al. , EMBO J. Homozygous disruption as described in 12,811-820 (1993), the endogenous mouse heavy chain gene is as described in Example 1 of WO 01/09187. It is homozygously destroyed. This mouse strain was described in Fishwild et al. , Nature Biotechnology 14,845-851 (1996), which carries the human kappa light chain transgene, KCo5. This mouse strain also carries a human heavy chain introduction chromosome composed of the chromosome 14 antigen binding fragment hCF (SC20), as described in WO 02/4348.

Spleen cells derived from these transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies according to well-known techniques. The human monoclonal or polyclonal antibody of the present invention, or an antibody of the present invention derived from another species, also produces and recovers another non-human mammalian or plant transgenic for the relevant immunoglobulin heavy and light chain sequences. It can be gene-introduced by the production of the antibody in a possible form. In connection with transgenic production in mammals, antibodies can be produced and recovered from goat, bovine, or other mammalian milk. See, for example, US Pat. No. 5,827,690, US Pat. No. 5,756,687, US Pat. No. 5,750,172 and US Pat. No. 5,741,957. I want to be.

The antibodies of the invention can be of any isotype. Isotype selection is typically guided by the desired effector function, such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. The human light chain constant region, either κ or λ, can be used. If desired, the class of anti-α-synuclein antibodies of the invention can be switched by known methods. For example, an antibody of the invention that was originally IgM can be class switched to an IgG antibody of the invention. In addition, class switching techniques can be used to convert one IgG subclass to another IgG subclass, for example from IgGl to IgG2. Thus, the effector function of the antibodies of the invention can be altered for a variety of therapeutic uses, for example, by isotype switching to IgG1, IgG2, IgG3 or IgG4 antibodies. In one embodiment, the antibody of the invention is an IgG1 antibody, such as IgG1, κ. An antibody is said to be of a particular isotype if its amino acid sequence is most homologous to that isotype compared to other isotypes.

In one embodiment, the antibody of the invention is a full length antibody, preferably an IgG antibody, in particular an IgG1, κ antibody. In another embodiment, the antibody of the invention is an antibody fragment or single chain antibody.

Antibodies and antigen-binding fragments thereof are obtained, for example, by antigen-binding fragmentation using conventional techniques, and antigen-binding fragments can be screened for usefulness as described herein for all antibodies. For example, an F (ab') 2 antigen binding fragment can be produced by treating the antibody with pepsin. The resulting F (ab') 2 antigen-binding fragment can be treated to reduce the disulfide bridge to produce a Fab'antigen-binding fragment. Fab antigen-binding fragments are obtained by treating IgG antibodies with papain; Fab'antigen binding fragments are obtained by pepsin digestion of IgG antibodies. The F (ab') antigen binding fragment can also be produced by binding the Fab'described below by a thioether bond or a disulfide bond. The Fab'antigen binding fragment is an antibody antigen binding fragment obtained by cleaving the disulfide bond in the hinge region of F (ab') 2. The Fab'-antigen binding fragment is obtained by treating the F (ab') 2 antigen binding fragment with a reducing agent such as dithiothreitol. Antibody-antigen-binding fragments can also be produced by expression of nucleic acids encoding such antigen-binding fragments in recombinant cells (see, eg, Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). .. For example, a chimeric gene encoding a portion of an F (ab') 2 antigen binding fragment is a DNA encoding the CH1 and hinge regions of the H chain to produce such a cleaved antibody antigen binding fragment molecule. It may contain a sequence followed by a translation stop codon.

In one embodiment, the anti-α-synuclein antibody is described in WO 2007059782 with a deletion of the monovalent antibody, preferably the hinge region, which is incorporated herein by reference in its entirety. It is a monovalent antibody. Thus, in one embodiment, the antibody is a monovalent antibody and the anti-α-sinucrane antibody is i) a step of providing a nucleic acid construct encoding the light chain of the monovalent antibody, wherein the construct is: The above comprising a nucleotide sequence encoding the VL region of the selected antigen-specific anti-α-synuclein antibody and a nucleotide sequence encoding the constant CL region of Ig, wherein the VL region of the selected antigen-specific antibody is encoded. The nucleotide sequence and the nucleotide sequence encoding the CL region of Ig are operably linked together, and for the IgG1 subtype, the nucleotide sequence encoding the CL region is in the presence of polyclonal human IgG or to an animal or human. With the step of modifying an amino acid capable of forming a disulfide bond or a covalent bond with another peptide containing the same amino acid sequence of the CL region so as not to contain the CL region when administered; ii). A step of providing a nucleic acid construct encoding a heavy chain of a valent antibody, wherein the construct comprises a nucleotide sequence encoding the VH region of the selected antigen-specific antibody and a nucleotide sequence encoding the constant CH region of human Ig. Containing, where the nucleotide sequence encoding the CH region is the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, are present in polyclonal human IgG. Amino acid residues involved in the formation of disulfide or covalent or stable non-covalent heavy chain bonds, together with other peptides containing the same amino acid sequence in the CH region of human Ig, when administered under or to animal humans. The nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operably linked together. Iii) By co-expressing the nucleic acid constructs of (i) and (ii) in the cells of the cell expression system of iv) (iii) with the step of providing the cell expression system to generate the monovalent antibody. , A method comprising the step of producing the monovalent antibody.

Similarly, in one embodiment, the anti-α-synuclein antibody is:
(I) A monovalent antibody comprising a variable region or an antigen-binding portion of the antibody of the invention described herein, and (ii) a CH region or a region thereof including CH2 and CH3 regions of an immunoglobulin. Here, the CH region or its region is the region corresponding to the hinge region and, if the immunoglobulin is not an IgG4 subtype, other regions of the CH region, such as the CH3 region, are identical in the presence of polyclonal human IgG. It is modified to be free of amino acid residues capable of forming a disulfide bond with the CH region or forming another covalent or stable non-covalent heavy chain bond with the same CH region.

In a further embodiment, the heavy chain of the monovalent anti-α-synuclein antibody is modified so that the entire hinge region is deleted.

In another further embodiment, the sequence of the monovalent antibody is modified so that it does not contain a receptor site for N-linked glycosylation.

The present invention is part of a bivalent or multivalent bispecific skeleton in which the anti-α-sinucrane binding region (eg, the α-sinucrane binding region of an anti-α-sinucrane monoclonal antibody) targets two or more epitopes. Also includes "bispecific antibodies" (eg, a second epitope such that the bispecific antibody may exhibit improved transcytosis that transcends biological barriers such as the blood-brain barrier. Can include epitopes of active transport receptors). Thus, in another further embodiment, monovalent Fabs of anti-synuclein antibodies can be bound to additional Fabs or scfvs that target different proteins to produce bispecific antibodies. Bispecific antibodies have dual functions, such as therapeutic functions provided by anti-synuclein binding regions, and transport functions that can bind to receptor molecules to facilitate transport across biological barriers such as the blood-brain barrier. May have.

The anti-α-synuclein antibody of the present invention and an antigen-binding fragment thereof also include a single-chain antibody. Single-chain antibodies are peptides to which the heavy and light chain Fv regions are bound. In one embodiment, the invention presents the heavy and light chains in Fv of the anti-α-sinucrane antibody of the invention with amino acid residues of about 10, 12, 15 or more in the peptide single chain (typically about 10, 12, 15 or more). It provides a single chain Fv (scFv) that is bound to the (based) flexible peptide linker. Methods for producing such antibodies are described, for example, in US Pat. No. 4,946,778, Packthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et al. , Science 242,423-426 (1988), Huston et al. , PNAS USA 85, 5879-5883 (1988) and McCafferty et al. , Nature 348, 552-554 (1990). Single-chain antibodies are monovalent when only a single VH and VL are used, divalent when two VHs and VLs are used, or used by three or more VHs and VLs. If so, it can be multivalued.

The anti-α-synuclein antibodies and antigen-binding fragments thereof described herein can be modified by inclusion of any suitable number of modified amino acids and / or binding to such conjugated substituents. Compatibility in this regard is generally determined by the ability to at least substantially retain α-synuclein selectivity and / or anti-α-synuclein specificity associated with the non-derivatized parental anti-synuclein antibody. Inclusion of one or more modified amino acids can be advantageous, for example, to increase polypeptide serum half-life, reduce polypeptide antigenicity, or increase polypeptide storage stability. Amino acids are, for example, modified at the same time as translation (co-translationally) or post-translational (post-translationally) during recombinant production (eg, N in the N-XS / T motif upon expression in mammalian cells). It is modified by (linked glycosylation) or synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, plenylated (eg, farnesylated, geranylgeranylated) amino acids, acetylated amino acids, acylated amino acids, PEGylated amino acids, biotinylated amino acids, carboxylated. Examples include amino acids and phosphorylated amino acids. There are sufficient references throughout the literature to guide those skilled in the art of amino acid modifications. An example protocol can be found in Walker (1998) Protein Protocols On CD-ROM, Humana Press, Totowa, NJ. The modified amino acid can be selected from, for example, glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to lipid moieties, or amino acids conjugated to organic inducers. ..

Anti-α-synuclein antibodies can also be chemically modified by covalent binding to polymers, eg, to increase their blood half-life. Exemplary polymers and methods for binding them to peptides are, for example, US Pat. No. 4,766,106, US Pat. No. 4,179,337, US Pat. No. 4,495,285. It is shown in the specification and US Pat. No. 4,609,546. Further exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (eg, about 1,000 to about 40,000, such as about 2,000 to about 20,000, such as about 3,000 to. PEG) having a molecular weight of 12,000 g / mol.

The antibodies of the invention can be further used in diagnostic methods or as ligands for diagnostic imaging.

In one embodiment, an anti-α-synuclein antibody comprising one or more radiolabeled amino acids is provided. Radiolabeled anti-α-synuclein antibodies can be used for both diagnostic and therapeutic purposes (binding to radiolabeled molecules is another possible feature). Non-limiting examples of such labels are, but are not limited to, bismuth ( ²¹³ Bi), carbon ( ¹¹ C, ¹³ C, ¹⁴ C), chromium ( ⁵¹ Cr), cobalt ( ⁵⁷ Co, ⁶⁰ Co). , Copper ( ⁶⁴ Cu), Ytterbium ( ¹⁶⁵ Dy), Erbium ( ¹⁶⁹ Er), Fluorine ( ¹⁸ F), Gadrinium ( ¹⁵³ Gd, ¹⁵⁹ Gd), Gallium ( ⁶⁸ Ga, ⁶⁷ Ga), Germanium ( ⁶⁸ Ge), Gold ( ¹⁹⁸ Au), Holmium ( ¹⁶⁶ Ho), Hydrogen ( ³ H), Indium ( ¹¹¹ In, ¹¹² In, ¹¹³ In, ¹¹⁵ In), Ytterbium ( ¹²¹ I, ¹²³ I, ¹²⁵ I, ¹³¹ I), Ytterbium ( ¹⁹² ) Ir), iron ( ⁵⁹ Fe), krypton ( ^{81 m} Kr), lantern ( ¹⁴⁰ La), lutetium ( ¹⁷⁷ Lu), manganese ( ⁵⁴ Mn), molybdenum ( ⁹⁹ Mo), nitrogen ( ¹³ N, ¹⁵ N), oxygen ( ¹⁵ O), Palladium ( ¹⁰³ Pd), Phosphorus ( ³² P), Potassium ( ⁴² K), Placeodium ( ¹⁴² Pr), Promethium ( ¹⁴⁹ Pm), Renium ( ¹⁸⁶ Re, ¹⁸⁸ Re), Rhodium ( ¹⁰⁵ Rh), Rubidium ( ⁸¹ Rb, ⁸² Rb), ruthenium ( ⁸² Ru, ⁹⁷ Ru), samarium ( ¹⁵³ Sm), scandium ( ⁴⁷ Sc), selenium ( ⁷⁵ Se), sodium ( ²⁴ Na), strontium ( ⁸⁵ Sr, ⁸⁹ Sr, ⁹² ) Sr), sulfur ( ³⁵ S), technetium ( ⁹⁹ Tc), talium ( ²⁰¹ Tl), tin ( ¹¹³ Sn, ¹¹⁷ Sn), xenon ( ¹³³ Xe), ytterbium ( ¹⁶⁹ Yb, ¹⁷⁵ Yb, ¹⁷⁷ Yb), ytterbium. ⁹⁰ Y) and zinc ( ⁶⁵ Zn). Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (eg, Junghans et al., Cancer Chemotherapy and Biotherapy 655-686 (2nd edition, Staffer patent, Longo, ed.). (1996)) and US Pat. No. 4,681,581, US Pat. No. 4,735,210, US Pat. No. 5,101,827, US Pat. No. 5,102,990. See US Reissue Patent No. 35,500, US Pat. No. 5,648,471 and US Pat. No. 5,697,902. For example, the radioactive isotope is chloramine. It can be combined by the T method (Lindegren, S. et al. (1998) "Choloramine-T InHigh-Specific-Activity Radioiodination Of Substance Biol.25 (7):. 659-665; Kurth, M.et al (1993) "Site-Specific Conjugation Of A Radioiodinated Phenethylamine Derivative To A Monoclonal Antibody Results In Increased Radioactivity Localization In Tumor", J.Med.Chem. 36 (9): 1255-1261; Rea, D.W. et al. (1990) "Site-specifically patented patenting tumors", Cancer Res.50 (3 Suppl): 857s-86.

The present invention relates to fluorescent labels (such as rare earth chelates (eg, europium chelate)), fluorescein-type labels (eg, fluorescein, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, dichlorotriazinylamine fluorescein), rhodamine-type labels. (For example, ALEXA FLUOR® 568 (Invitrogen), TAMRA® or Dansir Rhodamine), VIVOTAG 680 XL FLOUROCHROME® (Perkin Elmer), Phicoerythrin; Umberiferon, Lissamine; Cyanin; Also, anti-α-fluorescein antibodies and antigen-binding fragments thereof that are detectable detectable with BODIPY FL-SE® (Invitrogen) or their analogs (all of which are suitable for optical detection). offer. Chemiluminescent labels may be used (eg, luminol, luciferase, luciferin, and aequorin). Such diagnostics and detections also include, but are not limited to, the diagnostic molecules of the invention, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or enzymes including acetylcholinesterase. It can be done by binding to a detectable substance, or, but not limited to, a prosthetic group complex such as streptavidin / biotin and avidin / biotin.

Chemiluminescent labels may be used (eg, luminol, luciferase, luciferin, and aequorin). Such diagnostics and detections also include, but are not limited to, the diagnostic molecules of the invention, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or enzymes including acetylcholinesterase. It can be done by binding to a detectable substance, or, but not limited to, a prosthetic group complex such as streptavidin / biotin and avidin / biotin. Paramagnetic labels can also be used and are preferably detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT). Such paramagnetic labels are not limited, but are aluminum (Al), barium (Ba), calcium (Ca), terbium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), and gadolinium. (Gd), terbium (Ho), iridium (Ir), lithium (Li), magnesium (Mg), manganese (Mn), molybdenum (M), neodymium (Nd), osmium (Os), oxygen (O), palladium (Pd), Platinum (Pt), Rodium (Rh), Luthenium (Ru), Samalium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thurium (Tm), Tin (Sn), Titanium (Ti), tungsten (W), and zirconium (Zi), especially Co ⁺² , CR ⁺² , Cr ⁺³ , Cu ⁺² , Fe ⁺² , Fe ⁺³ , Ga ⁺³ , Mn ⁺³ , Ni ⁺² , Ti ⁺³ , V ⁺³ , And V ^{+ 4} paramagnetic ions, positron-emitting metals using various positron-emitting tomography methods, and compounds containing non-radiomagnetic paramagnetic metal ions.

Thus, in one embodiment, the anti-α-synuclein antibody of the invention can be labeled with a fluorescent label, chemiluminescent label, paramagnetic label, radioisotope label or enzyme label. Labeled antibodies can be used to detect or measure the presence or amount of said α-synuclein in a subject's brain. The method may include detection or measurement of in vivo imaging of the anti-α-synuclein antibody bound to the α-synuclein, and may include ex vivo imaging of the anti-α-synuclein antibody bound to the α-synuclein.

In a further aspect, the invention relates to an expression vector encoding one or more polypeptide chains of an antibody of the invention or an antigen-binding fragment thereof. Such expression vectors can be used for recombinant production of the antibodies and antigen binding fragments of the invention.

The expression vector in the context of the present invention can be any suitable DNA or RNA vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression control elements). Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the anti-α-synuclein antibody-encoding nucleic acid is described, for example, in a linear expression element (eg, as described in Vector Johnson, Nat Biotech 12,355-59 (1997)). Compressed nucleic acid vector (eg, as described in US Pat. No. 6,077,835 and / or WO 00/7807), pBR322, pUC 19/18, or pUC 118 /. 119, Naked DNA or RNA vectors containing plasmid vectors such as the "midge" minimum size nucleic acid vector (eg, as described in Schakowski et al., MoI Ther 3,793-800 (2001)). In, or in precipitate nucleic acid vector constructs such as CaPO ₄ precipitate constructs (eg, WO 00/46147, Benvenisty and Reshef, PNAS USA 83,9551-55 (1986), Willer et al., Cell 14,725 (eg). 1978), and as described in Coraro and Pearson, Somatic Cell Genetics 2,603 (1981)). Such nucleic acid vectors and their uses are well known in the art (see, eg, US Pat. No. 5,589,466 and US Pat. No. 5,973,972).

In one embodiment, the vector is suitable for expression of an anti-α-synuclein antibody or antigen-binding fragment thereof in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vector (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989), pET vector (Novagen, Madison, WI), etc.). Can be mentioned.

The expression vector can further or instead be a suitable vector for expression in a yeast system. Any vector suitable for expression in the yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase and PGH (F. Ausubel et al., Ed. Current Protocols in Molecular Biology, Greene 1987 New York (1987), Grant et al., Methods in Enzymol 153,516-544 (1987), Mattanovich, D. et al. Methods Mol. Biol. 824, 329-358 (2012), Celik, E. et al. Biotechnol.Adv.30 (5), 11081-118 (2012), Li, P. et al. Appl. Biochem. Biotechnol. 142 (2), 105-124 (2007), Boeer, E. et al. Appl. Microbiol. Biotechnol. 77 (3), 513-523 (2007), van der Vaart, JM Methods Mol. Biol. 178, 359-366 (2002), and Holliger, P. Methods Mol. Biol. , 349-357 (2002)).

In the expression vector of the invention, the anti-α-synuclein antibody-encoding nucleic acid may contain or be bound to any suitable promoter, enhancer, and other expression-promoting element. Examples of such elements include potent expression promoters (eg, human CMV IE promoter / enhancer and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, E. coli ( The origin of replication of the plasmid product in E. coli), the antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (eg, a polylinker). Nucleic acids can also include inducible promoters as opposed to constitutive promoters such as CMVIE (those skilled in the art will appreciate that such terms are in fact a descriptive term for the degree of gene expression under certain conditions. Will recognize).

Antibodies to the antigen-binding fragments of the invention can be obtained from various cell lines such as human cell lines, non-human mammalian cell lines, and insect cell lines such as CHO cell lines, HEK cell lines, BHK-21 cell lines, mouse cells. Strains (myeloma cell lines, etc.), fibrosarcoma cell lines, PER. It can be produced in C6 cell lines, HKB-11 cell lines, CAP cell lines and HuH-7 human cell lines (contents are incorporated herein by reference, Dumont et al, 2015, Crit Rev Biotechnol. Sep 18: 1-13.).

In yet another embodiment, the invention is a transfectoma that produces an antibody of the invention as defined herein or an antigen binding region thereof or a bispecific molecule of the invention as defined herein. For recombinant eukaryotic or prokaryotic host cells, such as transfectoma). Examples of host cells include yeast, bacteria, and mammalian cells (such as CHO or HEK cells). For example, in one embodiment, the invention provides a cell comprising a nucleic acid stably integrated into a cellular genome comprising a sequence code for expression of the anti-α-synuclein antibody of the invention or an antigen-binding fragment thereof. In another embodiment, the invention comprises cells comprising an unfused nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising the sequence code for expression of the anti-α-synuclein antibody of the invention. offer.

In a further embodiment, the invention is a method for producing the anti-α-synuclein antibody of the invention, a) the step of culturing the hybridoma or host cell of the invention as described herein above. b) The present invention relates to a method comprising a step of purifying the antibody of the present invention from a medium.

In one embodiment, the invention is a preparation that, when such a term is used herein, comprises an anti-α-synuclein antibody as defined herein and binds to α-synuclein. It relates to a preparation that is substantially free of naturally occurring antibodies that is not possible or does not substantially alter the anti-α-synuclein functionality of the preparation. Thus, such preparations do not include naturally occurring serum, or purified derivatives of such serum, and another anti-α-synuclein antibody and another that does not alter the functionality of the preparation's anti-α-synuclein antibody. Containing mixtures with antibodies; where such functionality is
(I) Binding affinity of anti-α-synuclein antibody for α-synuclein (KD);
(Ii) Ability of anti-α-synuclein antibody to inhibit protease cleavage of α-synuclein fibrils;
(Iii) Ability of anti-α-synuclein antibody to ameliorate impaired basal synaptic transmission in F28-snca transgenic mice;
(Iv) Ability of anti-α-synuclein antibody to reduce levels of α-synuclein in mouse hippocampus as measured by in vivo microdialysis; and (v) in a rat model of Parkinson's disease when administered chronically. Ability of anti-α-synuclein antibody to restore motor function,
(Vi) Ability to prevent seeding of α-synuclein (such as in vitro and / or accumulation of insoluble phosphorylated α-synuclein in a mouse model of Parkinson's disease); and / or (vi) cleavage α-synuclein in the human brain Selected from the group consisting of the ability to bind to.

The present invention particularly has such anti-synuclein having structural changes in its amino acid sequence (either in its CDR, variable region, framework residue and / or constant region) compared to the structure of the native anti-α-synuclein antibody. A preparation of an α-synuclein antibody, wherein the structural changes significantly alter (ie, functional) the functionality of the anti-α-synuclein monoclonal antibody compared to the functionality exhibited by the native anti-α-synuclein antibody. Over 20% difference, over 40% difference, over 60% difference, over 80% difference, over 100% difference, over 150% difference, over 2x difference, over 4x difference, 5x Super-difference, or more than 10-fold difference); where such functionality is
(I) Binding affinity of anti-α-synuclein monoclonal antibody for α-synuclein (KD);
(Ii) Ability of anti-α-synuclein monoclonal antibody to inhibit protease cleavage of α-synuclein fibrils;
(Iii) Ability of anti-α-synuclein monoclonal antibody to ameliorate impaired basal synaptic transmission in F28-snca transgenic mice;
(Iv) Ability of anti-α-synuclein monoclonal antibody to reduce levels of α-synuclein in mouse hippocampus as measured by in vivo microdialysis; and / or (v) Parkinson's disease when administered chronically Ability of anti-α-synuclein monoclonal antibody to restore motor function in rat model;
(Vi) Ability to prevent seeding of α-synuclein (such as in vitro and / or accumulation of insoluble phosphorylated α-synuclein in a mouse model of Parkinson's disease); and / or (vi) cleavage α-synuclein in the human brain The ability to bind to, in particular, such altered functionality is the result of structural changes and is therefore inseparable from it.

The term "substantially free" of naturally occurring antibodies is such a preparation that does not substantially alter the α-synuclein binding properties of such naturally occurring antibodies or of such preparations. Refers to the complete absence of inclusion of such naturally occurring antibodies at certain concentrations in an object. An antibody is said to be "isolated" if it has no naturally occurring counterpart or if it has been separated or purified from a naturally occurring component.

The term "spontaneous antibody", when it comes to such preparations, is an antibody that is induced in living humans or other animals as a natural result of the functioning of their immune system (spontaneous autoantibodies). (Including antibodies).

Accordingly, the preparations of the invention do not exclude anti-α-synuclein antibodies and such preparations containing additional antibodies that are intentionally added capable of binding to epitopes not retained by α-synuclein. In fact it explicitly embraces it. Such preparations, in particular, include Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gauche's disease, diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBV). ), Includes embodiments thereof showing improved efficacy in treating synucleinopathy such as combined Alzheimer's disease and Parkinson's disease, pure autonomic failure and multiple system atrophy.

In yet another aspect, the invention is described.
(I) Anti-α-synuclein antibody or antigen-binding fragment thereof, or preparation thereof as defined herein, such anti-α-synuclein antibody as such terminology is defined herein. Or a preparation comprising an antigen-binding fragment thereof, and (ii) a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

Pharmaceutical compositions are described in Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Ed. , Mac Publishing Co., Ltd. , Easton, PA, according to prior art such as those disclosed in 2013, may be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuncts and excipients.

Pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the selected compounds and methods of administration of the invention. Compatibility for carriers and other ingredients of a pharmaceutical composition is determined based on the absence of significant adverse effects on the desired biological properties of the selected compound or pharmaceutical composition of the invention (eg, epitopes). Not significant effect on binding (less than 10% relative inhibition, 5% or less relative inhibition, etc.).

The pharmaceutical composition of the present invention comprises diluents, fillers, salts, buffers, detergents (eg, nonionic detergents such as Tween-20 or Tween-80), stabilizers (eg, sugars or proteins). No amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in pharmaceutical compositions may also be included. Diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate buffered saline, Ringer's solution, dextrose solution, and Hanks solution. In addition, the pharmaceutical composition or formulation may also include other carriers, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. The compositions include proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (eg, latex functionalized sepharose, agarose, cellulose, etc.), polymer amino acids, amino acid copolymers, and lipid aggregates (eg, latex functionalized). It can also include large, slowly metabolized polymers (eg, oil droplets or liposomes).

The actual dose level of the active ingredient in the pharmaceutical composition of the invention is varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response to a particular patient, composition, and method of administration. obtain. The dose level selected is combined with the activity of the particular composition of the invention used, or its amide, route of administration, time of administration, rate of excretion of the particular compound used, duration of treatment, specific composition used. Various drugs including other drugs, compounds and / or materials used in the treatment, age, sex, weight, pathology, overall health and past medical history of the patient being treated, and similar factors well known in the medical field. It depends on the dynamic factors.

The pharmaceutical composition may be administered by any suitable route and method, including parenteral, topical, oral or nasal means for prophylactic and / or therapeutic treatment. In one embodiment, the pharmaceutical composition of the invention is administered parenterally. As used herein, the terms "parenteral administration" and "administered parenterally" usually refer to methods of administration other than injectable, intestinal and topical administration, epidermal, intravenous. Intramuscular, intraarterial, intrathecal, intrasacular, intraocular, intracardiac, intracutaneous, intraperitoneal, intratenal, transtracheal, subcutaneous, subepithelial, intra-articular, subcapsular, subepidal, intraspinal, intracranial Includes intra-, intrathoracic, epidural and intrathoracic injections and infusions. Further suitable routes for administering the compounds of the invention in vivo and in vitro are well known in the art and can be selected by one of ordinary skill in the art. In one embodiment, the pharmaceutical composition is administered intravenously or by subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption retarders that are physiologically compatible with the compounds of the invention. Can be mentioned.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, physiological saline, phosphate buffered saline, ethanol, dextrose, polyols (glycerol, propylene glycol, polyethylene glycol, etc.). , And suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacant gum and injectable organic acid esters (such as ethyl oleate), and / or various buffers. Liquid is mentioned. Other carriers are well known in the pharmaceutical field.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in the pharmaceutical compositions of the present invention is envisioned, unless conventional vehicles or agents are incompatible with the active compound.

Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, in the case of dispersions by maintaining the required particle size, and by the use of surfactants.

The pharmaceutical composition of the present invention comprises pharmaceutically acceptable antioxidants such as (1) water-soluble antioxidants (ascorbic acid, cysteine hydrochloride, sodium bicarbonate, sodium metabisulfite, sodium sulfite, etc.); (2). ) Oil-soluble antioxidants (ascorbic palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.); and (3) metal chelating agents (citrate, It may also contain ethylenedidiamine tetraacetic acid (EDTA), sorbitol, tartrate acid, phosphoric acid, etc.).

The pharmaceutical composition of the present invention may also contain isotonic agents such as sugars, polyalcohols (mannitol, sorbitol, glycerol, etc.) or sodium chloride in the composition.

The pharmaceutical compositions of the present invention are suitable for the route of administration of choice, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which can improve the shelf life or effectiveness of the pharmaceutical composition. It may also contain one or more adjuncts. The compounds of the invention can be prepared with carriers that protect the compound from immediate release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. It may be included alone or with wax or other materials known in the art. Methods for preparing such formulations are generally known to those of skill in the art. For example, Sustained and Controlled Relax Delivery Systems, J. Mol. R. Robinson, ed. , Marcel Dekker, Inc. , New York, 1978.

In one embodiment, the compounds of the invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in the pharmaceutical compositions of the present invention is envisioned, unless conventional vehicles or agents are incompatible with the active compound. Auxiliary active compounds can also be incorporated into the composition.

Pharmaceutical compositions for injection typically must be sterile and stable under conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic acid esters (such as ethyl oleate). It can be an aqueous or non-aqueous solvent or dispersion medium. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, in the case of dispersions by maintaining the required particle size, and by the use of surfactants. In many cases, it will be preferred to include isotonic agents, such as sugars, polyalcohols (glycerol, mannitol, sorbitol, etc.), or sodium chloride in the composition. Sustained absorption of the injectable composition can be achieved by including in the composition substances that delay the absorption of the antibody, such as monostearate and gelatin. Sterilized injection solutions can be prepared by incorporating the required amount of active compound in a suitable solvent containing, for example, one or a combination of components as listed above, and if necessary, subsequent sterile microfiltration. .. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from, for example, the components listed above. For sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum-dried and freeze-dried (freeze-dried) to obtain powders of the active ingredient and any additional desired ingredients from those solutions that have been sterile filtered. ).

Sterile injectable solutions can be prepared by incorporating the required amount of active compound in a suitable solvent containing one or a combination of components as listed above and, if necessary, subsequent sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from the components listed above. For sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum-dried and freeze-dried (freeze-dried) to obtain powders of the active ingredient and any additional desired ingredients from those solutions that have been sterile filtered. ).

The above methods of treatment and the dosing regimen in the uses described herein are tailored to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, some divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the treatment situation. May be good. The parenteral composition can be formulated in a unit dosage form for ease of administration and uniformity of administration. Unit dosage forms as used herein refer to physically separate units suitable as a unified dose for the subject being treated; each unit is associated with the required pharmaceutical carrier. It contains a predetermined amount of active compound calculated to produce the desired therapeutic effect. The specification of the unit dosage form of the present invention is that (a) the unique properties of the active compound and the specific therapeutic effect obtained, and (b) the treatment of such active compound in an individual. It is subject to and directly dependent on the limitations specific to the technical field of formulation.

Effective doses and dosing regimens of anti-α-synuclein antibodies will depend on the disease or condition being treated and may be determined by one of ordinary skill in the art. Predetermined doses are administered on any day, and the doses can range from about 0.0001 to about 100 mg / kg, more usually from about 0.01 to about 5 mg / kg host body weight. For example, the dose can be in the range of 1 mg / kg body weight or 10 mg / kg body weight or 1-10 mg / kg body weight. Therefore, exemplary doses include about 0.1 to about 10 mg / kg / body weight, about 0.1 to about 5 mg / kg / body weight, about 0.1 to about 2 mg / kg / body weight, and about 0.1. ~ About 1 mg / kg / body weight, for example, about 0.15 mg / kg / body weight, about 0.2 mg / kg / body weight, about 0.5 mg / kg / body weight, about 1 mg / kg / body weight, about 1.5 mg / kg / body weight. Examples include body weight, about 2 mg / kg / body weight, about 5 mg / kg / body weight, or about 10 mg / kg / body weight.

Physicians with conventional skills in the art can easily determine and prescribe effective amounts of the required pharmaceutical composition. For example, physicians may start with a dose of anti-α-synuclein antibody used in a pharmaceutical composition at a lower level than required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Can increase to. In general, a suitable daily dose of the composition of the invention would be the amount of compound that is the lowest effective dose to produce a therapeutic effect. Such effective doses generally depend on the factors mentioned above. Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous administration. If necessary, the effective daily dose of the pharmaceutical composition is optionally in unit dosage form, administered separately at appropriate intervals throughout the day, 2, 3, 4, 5, 6 It can be administered in divided doses of multiple doses or more. The compound of the present invention can be administered alone, but it is preferable to administer the compound as a pharmaceutical composition as described above.

Labeled antibodies of the invention can be used for diagnostic purposes to detect, diagnose, or monitor a disease or disorder. The present invention is, but is not limited to, Parkinson's disease, idiopathic Parkinson's disease, familial Parkinson's disease, diffuse Levy body disease (DLBD), Levy body variant Alzheimer's disease (LBV), combined Alzheimer's disease and Parkinson's. Provides detection or diagnosis of neurodegenerative or cognitive disorders or disorders, including disease, pure autonomic neuropathy or multilineage atrophy, with (a) one or more antibodies that specifically bind to α-sinucrane. In the step of measuring the presence of α-sinucrane species and fragments in a subject's cell or tissue sample; (b) the level of the antigen is compared to a control level, eg, a level in a normal tissue sample, thereby thereby An increase in the measured level of the antigen compared to the control level of the antigen comprises the steps of indicating the disease or disorder or the severity of the disease or disorder.

The antibodies of the invention can be used to measure α-synuclein monomers, oligomers, fibrous morphologies or fragments of α-synuclein in biological samples using immunohistochemical methods well known in the art. .. Other antibody-based methods useful for detecting proteins include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and mesoscale discovery platform-based assay (MSD). Immunoassay method is mentioned. Suitable antibody labels can be used in such kits and methods, and known labels in the art include enzyme labels (such as alkaline phosphatase and glucose oxidase); radioisotope labels (iodine ( ¹²⁵ I, ¹³¹ I). ), Carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In), and technetium ( ^{99 m} Tc); and luminescent labels (such as luminol and luciferase); and fluorescent labels (fluorescein). And Rhodamine, etc.).

The presence of labeled anti-α-synuclein antibodies or their α-synuclein binding fragments can be detected in vivo for diagnostic purposes. In one embodiment, the diagnosis is a) the step of administering to the subject an effective amount of such a labeled molecule; b) after administration, waiting for a predetermined time interval to place the labeled molecule at the site of Aβ deposition (if). (If any) enriched to allow the unbound labeled molecule to be removed until it reaches the background level; c) the step of determining the background level; and d) the labeled molecule above the background level. Detection comprises the step of detecting a labeled molecule in a subject such that the subject suffers from a disease or disorder or indicates the severity of the disease or disorder. According to such an embodiment, the molecule is labeled with an imaging moiety suitable for detection using a particular imaging system known to those of skill in the art. Background levels can be determined by a variety of methods known in the art, including comparing the amount of labeled antibody detected to standard values predetermined for a particular imaging system. Methods and systems that can be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and the like. And ultrasonography.

In a further aspect, the invention relates to an antibody of the invention or an antigen-binding fragment thereof for medical use.

In a further aspect, the invention relates to an antibody of the invention or an antigen-binding fragment thereof for use in treating, diagnosing or imaging synucleinopathy.

In one embodiment, the monoclonal antibody, or antigen-binding fragment thereof, is Parkinson's disease, idiopathic Parkinson's disease, familial Parkinson's disease, diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBV), complex. It is intended for use in the treatment of Alzheimer's disease and Parkinson's disease, pure autonomic failure or multiple system atrophy.

In a further aspect, the invention relates to the use of the antibodies of the invention, or antigen-binding fragments thereof, in the manufacture of agents for treating, diagnosing or imaging synucleinopathy.

In a further aspect, the invention relates to the treatment, diagnosis or imaging of Parkinson's disease or other synucleinopathy, comprising administering an effective dose of an antibody of the invention, or an antigen-binding fragment thereof.

Preferably, in the use and method of those embodiments of the invention, the treatment is long-term, preferably over at least 2 weeks, eg at least 1 month, 6 months, 1 year or more.

In a further aspect, the invention provides a kit comprising the antibody of the invention, or an antigen binding fragment thereof.

Embodiments of the present invention As will be apparent from the text and examples, the present invention further relates to the following embodiments.

1. 1. A monoclonal antibody capable of specifically binding to an epitope within amino acids 112 to 117 (SEQ ID NO: 9 (ILEDMP)) in α-synuclein, or an antigen-binding fragment thereof.

2. 2. The monoclonal antibody according to Embodiment 1, or an antigen-binding fragment thereof, which competes with antibody GM37 for binding to the epitope.

3. 3. The monoclonal antibody according to Embodiment 1, which is GM37, GM37 variant 1, GM37 variant 2 or GM37 variant 3, or an antigen-binding fragment thereof.

4. A monoclonal antibody capable of specifically binding to an epitope in amino acids 112 to 115 (SEQ ID NO: 19 (ILED)) in α-synuclein, or an antigen-binding fragment thereof.

5. GM285, the monoclonal antibody according to embodiment 1 or 4, or an antigen-binding fragment thereof.

6. The monoclonal antibody according to the preceding embodiment, or an antigen-binding fragment thereof, wherein the antibody comprises or comprises an intact antibody.

7. Fv fragments (eg, single-stranded Fv and disulfide-bound Fv), Fab-like fragments (eg, Fab fragment, Fab'fragment and F (ab) ₂ fragment) and domain antibodies (eg, single _VH variable region or _VL variable region). The monoclonal antibody according to any one of the preceding embodiments, which comprises or comprises an antigen-binding fragment selected from the group consisting of), or an antigen-binding fragment thereof.

8. The monoclonal antibody according to any one of the preceding embodiments, or an antigen-binding fragment thereof, wherein the monoclonal antibody is selected from the group consisting of antibodies of subtypes IgG1, IgG2, IgG3 and IgG4.

9. The antibody or antigen-binding fragment has the following characteristics:
(I) Binding affinity ( _KD ) for 0.5-10 nM α-synuclein, such as 1-5 nM or 1-2 nM;
(Ii) Ability to inhibit protease cleavage of α-synuclein fibrils;
(Iii) Ability to ameliorate impaired basal synaptic transmission in F28-snca transgenic mice;
(Iv) Ability to reduce levels of α-synuclein in mouse hippocampus as measured by in vivo microdialysis;
(V) Ability to restore motor function in a rat model of Parkinson's disease when administered chronically;
(Vi) Ability to prevent seeding of α-synuclein (such as in vitro and / or accumulation of insoluble phosphorylated α-synuclein in a mouse model of Parkinson's disease); and / or (vii) to cleavage α-synuclein in the human brain The monoclonal antibody, or antigen-binding fragment thereof, according to any one of the preceding embodiments, which exhibits one or more of the ability to bind.

10. The monoclonal antibody according to any one of the preceding embodiments, which is a human or humanized antibody, or an antigen-binding fragment thereof.

11. The following CDR:
a) GFTFSSYAMT (SEQ ID NO: 1) or an amino acid sequence having 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences;
b) AIRS (N / S / Q / H) GDRTD YADSVKG (SEQ ID NO: 2, 33, 34, 35) or 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, or Amino acid sequences with one or less amino acid differences; or c) AKNWAPFDS (SEQ ID NO: 3) or four or less amino acid differences, or three or less amino acid differences, or two or less amino acid differences, or one or less amino acids The monoclonal antibody or the monoclonal antibody according to Embodiments 1 to 3 and 6 to 10, or an antigen-binding fragment thereof, which comprises a heavy chain variable region containing an amino acid sequence having a difference.

12. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 11, comprising a heavy chain variable region comprising CDRs of SEQ ID NOs: 1 and 3 and one of SEQ ID NOs: 2 and 33, 34 or 35.

からなる群から選択される重鎖可変領域を含むかまたはそれからなる、実施形態１１に記載のモノクローナル抗体またはその抗原結合フラグメント。 13.

The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 11, which comprises or comprises a heavy chain variable region selected from the group consisting of.

14. The following CDR:
a) ASQSVSSSYLA (SEQ ID NO: 4) or an amino acid sequence having 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences;
b) GASSRAT (SEQ ID NO: 5) or an amino acid sequence having 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences; or c) QQYGSSPWT (sequence). No. 6) or a monoclonal antibody or a monoclonal antibody containing a light chain variable region containing an amino acid sequence having an amino acid difference of 4 or less, or an amino acid difference of 3 or less, or an amino acid difference of 2 or less, or an amino acid difference of 1 or less. The monoclonal antibody according to Embodiments 1 to 3 and 6 to 13, or an antigen-binding fragment thereof.

15. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 14, which comprises a light chain variable region comprising the CDRs of SEQ ID NOs: 4, 5 and 6.

を含むかまたはそれからなる軽鎖可変領域を含む、実施形態１４に記載の抗体またはその抗原結合フラグメント。 16. Amino acid sequence:

The antibody or antigen-binding fragment thereof according to embodiment 14, comprising or comprising a light chain variable region comprising or comprising.

を含むかまたはそれからなる軽鎖を含む、実施形態１４に記載のモノクローナル抗体またはその抗原結合フラグメント。 17. Amino acid sequence:

14. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 14, comprising a light chain comprising or comprising.

18. Embodiments 1 to include a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region comprising or comprising the amino acid represented by any of SEQ ID NOs: 7, 33, 34 or 35. 3 and the monoclonal antibody or antigen-binding fragment thereof according to 6 to 17.

19. Has increased thermal stability, such as increased stability to prevent aggregation or unfolding, as shown in FIG. 27, 2% -10% higher at temperatures above 65 ° C. compared to GM 37 wt. Stability is 2% to 8% higher than GM37 wt at temperatures above 65 ° C, or 2% to 5% higher than GM37 wt at temperatures above 65 ° C. The monoclonals according to embodiments 1-3 and 6-18, comprising a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region comprising or consisting of the amino acid set forth in SEQ ID NO: 34. An antibody or its antigen-binding fragment.

20. The following CDR:
a) AASGFTFSRFTMT (SEQ ID NO: 20) or an amino acid sequence having 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences;
b) AISGSGGGTS YADSVKG (SEQ ID NO: 21) or an amino acid sequence having 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences; or c) AKNWAPFDY ( SEQ ID NO: 22) or a monoclonal antibody comprising a heavy chain variable region comprising an amino acid sequence having 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences. Alternatively, the monoclonal antibody according to Embodiments 1 to 10, or an antigen-binding fragment thereof.

21. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 20, which comprises a heavy chain variable region comprising the CDRs of SEQ ID NOs: 20, 21 and 22.

を含むかまたはそれからなる重鎖可変領域を含む、実施形態２０に記載のモノクローナル抗体またはその抗原結合フラグメント。 22. Amino acid sequence

20. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 20, which comprises or comprises a heavy chain variable region comprising or comprising.

23. The following CDR:
d) RASQSVSRSSYLA (SEQ ID NO: 23) or an amino acid sequence having 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences;
e) GASSRAT (SEQ ID NO: 24) or an amino acid sequence having 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences; or f) QQYGSSPWT (sequence). No. 25) or a monoclonal antibody or a monoclonal antibody containing a light chain variable region containing an amino acid sequence having an amino acid difference of 4 or less, or an amino acid difference of 3 or less, or an amino acid difference of 2 or less, or an amino acid difference of 1 or less. The monoclonal antibody according to any one of Embodiments 1 to 10 and 20 to 22, or an antigen-binding fragment thereof.

24. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 23, which comprises a light chain variable region comprising the CDRs of SEQ ID NOs: 23, 24 and 25.

のアミノ酸配列を含むかまたはそれからなる軽鎖可変領域を含む、実施形態２４に記載の抗体またはその抗原結合フラグメント。 25.

24. The antibody or antigen-binding fragment thereof according to embodiment 24, which comprises or comprises a light chain variable region comprising or consisting of an amino acid sequence of.

26. To any one of the preceding embodiments comprising or comprising the amino acid sequence of SEQ ID NO: 27 or comprising the light chain variable region comprising any of SEQ ID NO: 26 and comprising the heavy chain variable region comprising or comprising the amino acid set forth in any of SEQ ID NO: 26. The monoclonal antibody or antigen-binding fragment thereof described.

27. The monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, comprising the Fc region.

28. The monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, further comprising a portion for increasing the in vivo half-life of the substance.

29. 13. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 28, wherein the moiety for increasing the in vivo half-life is selected from the group consisting of polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids and dextran. ..

30. The monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody polypeptide further comprises a detectable moiety.

31. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 30, wherein the detectable moiety is a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label or an enzyme label.

32. 13. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 30 or 31, wherein the detectable moiety comprises or comprises a radioisotope.

33. 13. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 32, wherein the radioisotope is selected from the group consisting of ^{99 m} Tc, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁸⁹ Zr, ¹²³ I and ²⁰¹ Tl.

34. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 30, wherein the detectable moiety comprises or consists of a paramagnetic isotope.

35. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 34, wherein the paramagnetic isotope is selected from the group consisting of ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr and ⁵⁶ Fe.

36. The monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 30 to 35, wherein the detectable moiety is detectable by imaging techniques such as SPECT, PET, MRI, optical or ultrasonic imaging.

37. The monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 30 to 36, wherein the detectable moiety is indirectly bound to the antibody or antigen-binding fragment thereof via a binding moiety.

38. The binding moiety is 1,4,7,10-tetraazacyclododecane-1,4,7,10, a derivative of tetraacetic acid (DOTA), deferroxamine (DFO), a derivative of diethylenetriaminepentaacetic acid (DTPA), S-2. -(4-Isotiocyanatobenzyl) -1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivative and 1,4,8,11-tetraazacyclododecane-1, 13. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 37, which is selected from the group consisting of derivatives of 4,8,11-tetraacetic acid (TETA).

39. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of the preceding embodiments or a component polypeptide chain thereof.

40. The nucleic acid molecule according to embodiment 39, wherein the molecule is a cDNA molecule.

41. 13. The nucleic acid molecule of embodiment 30 or 31, which encodes an antibody heavy chain or a variable region thereof.

42. The nucleic acid molecule according to any one of embodiments 39-41, which encodes an antibody light chain or a variable region thereof.

43. A vector containing the nucleic acid molecule according to any one of embodiments 39 to 42.

44. A recombinant host cell comprising the nucleic acid molecule according to any one of embodiments 39-42 or the vector according to embodiment 43.

45. Embodiment 44, which is a method for producing the antibody or antigen-binding fragment according to any one of embodiments 1 to 27, under conditions that allow expression of the encoded antibody or antigen-binding fragment thereof. A method comprising the step of culturing a host cell according to.

46. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment according to any one of embodiments 1 to 35 and a pharmaceutically acceptable carrier.

47. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 1-35 for medical use.

48. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 1-35 for use in treating, diagnosing or imaging synucleinopathy.

49. Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gauche disease, diffuse Lewy body disease (DLBD), Lewy body dementias Alzheimer's disease (LBV), combined Alzheimer's disease and Parkinson's disease, pure autonomic failure The monoclonal antibody or antigen-binding fragment thereof according to embodiment 48 for use in treating neuropathy and multiple system atrophy.

50. Use of the monoclonal antibody or antigen-binding fragment thereof according to Embodiment 1-35 in the manufacture of a drug for treating, diagnosing or imaging synucleinopathy.

51. Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gauche disease, diffuse Lewy body disease (DLBD), Lewy body dementias Alzheimer's disease (LBV), combined Alzheimer's disease and Parkinson's disease, pure autonomic failure Use of the monoclonal antibody or antigen-binding fragment thereof according to embodiment 50 in the manufacture of a drug for treating neuropathy and multiple system atrophy.

52. A method for treating, diagnosing, or imaging synucleinopathy in a subject, comprising the step of administering the pharmaceutical composition according to embodiment 46 to the subject in an effective amount.

53. Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gauche disease, diffuse Lewy body disease (DLBD), Lewy body dementias Alzheimer's disease (LBV), combined Alzheimer's disease and Parkinson's disease, pure autonomic failure The antibody for use according to embodiment 48, or an antigen-binding fragment thereof, or the use according to embodiment 50, or the method according to embodiment 52, for treating neuropathy and multiple system atrophy.

54. The antibody, or antigen-binding fragment thereof, for use according to embodiment 52 or 53, wherein the treatment is long-term; or use; or method.

55. The antibody, or antigen-binding fragment thereof, for use according to embodiment 52, wherein the long-term treatment spans at least 2 weeks, eg, at least 1 month, 6 months, 1 year or more; or use; or method.

56. The antibody or antigen-binding fragment thereof for use according to any one of embodiments 47-55, wherein the subject is a human; or use; or method.

57. A kit comprising the antibody according to Embodiments 1-35 or an antigen-binding fragment thereof.

58. The kit according to embodiment 57 for medical use.

59. 13. The monoclonal antibody or antigen-binding fragment thereof according to embodiments 30-35 for use in detecting or measuring the presence or amount of the α-synuclein in a subject's brain or body fluid.

60. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 59, wherein the detection or measurement comprises in vivo imaging of the anti-synuclein antibody bound to the α-synuclein.

61. 13. The monoclonal antibody or antigen-binding fragment thereof according to embodiments 30-35, wherein the detection or measurement comprises exvivo imaging of the anti-synuclein antibody bound to the α-synuclein.

Example 1: Antibody screening 1. Immunogen and Ligand Production The following proteins were obtained or produced for use as the immunogen shown in FIG. Mice were immunized with three immunogens: full-length recombinant human α-synuclein fibrils; human α-synuclein recombinant proteins containing amino acids 1-60 (Rpeptide, Bogart, Georgia) and human α containing amino acids 1-119. -Synuclein recombinant protein. A lyophilized product (Cat. No. S-1001-2) manufactured by Rpeptide (Bogart, Georgia) was used to generate fibrils from full-length α-synuclein. It was dissolved in 20 mM Tris and 300 mM NaCl buffer at a protein concentration of 1 mg / ml. Proteins in 96-well plates containing 70 μm diameter ceramic beads in each well at 200 rpm in a Voltem 56 Shaker Incubator (Labnet International, Edison, NJ, USA) at 37 ° C. for 7 days to make fibrils. The solution was incubated with 170 μl aliquots, after fibril formation, thioflavin T was added and fluorescence in one of the wells was measured. Recombinant α-synuclein containing amino acids 1-60 was dissolved in water to give a concentration of 1 mg / ml.

を、Ｇｅｎｓｃｒｉｐｔによって合成し、ｐＥＴ２４ａ（＋）発現ベクター（Ｎｏｖａｇｅｎ）におけるＮｄｅＩ－ＸｈｏＩ部位へとクローニングした。 Recombinant α-synuclein containing amino acids 1-119 was made using the following construct: a synthetic gene encoding a six amino acid histidine tag, followed by a factor Xa cleavage site and human α-synuclein amino acids 1-119. Array to code:

Was synthesized by Genscript and cloned into the NdeI-XhoI site in the pET24a (+) expression vector (Novagen).

The expression vector was transformed into E. coli BL21 and a single colony was harvested for expression using Novagen's overnight express self-induction system (User protocol TB383 rev. H1005). The scale was a final culture volume of 500 ml. Cells were harvested by centrifugation at 6000 g for 10 minutes and subsequently lysed with Bug Buster protein extraction reagent (User protocol TB245 Rev. 0304). After lysis, the sample was removed by centrifugation and the supernatant was used for further purification.

The His-tagged protein was purified on a 5 ml HisTrap column (GE healthcare) equilibrated in 20 mM sodium phosphate pH 7.5, 1 M NaCl (A-buffer). After application of the sample and washing with A-buffer, the protein was eluted over 20 column volumes to a gradient of 0.25 M imidazole in A-buffer. A 5 ml fraction was collected and analyzed by SDS-PAGE. Fractions containing the protein of interest were pooled, concentrated and applied to S200 (26/60) size exclusion columns (GE healthcare) in 10 mM Tris pH 7.4, 300 mM NaCl. Again, the fractions were pooled according to the presence of bands with the predicted size in SDS-PAGE.

To remove the N-terminal tag, purified his-tagged α-synucleins 1-119 were incubated with Factor Xa in a 1:50 distribution using the Novagen kit (69037-3FRX). After overnight incubation, factor Xa was removed in batches with Xarrest agarose. The cleaved α-synucleins 1-119 were finally purified by permissive HisTrap chromatography as described above. Purified α-synucleins 1-119 were obtained from the flow throttle and concentrated to about 400 μg / ml using a centricon concentrator.

Alpha-synuclein (Rpeptide) was rehydrated in PBS at 2 mg / ml and peroxynitilite (100 μL / mg protein) was added dropwise with mixing. The nitrosynylated α-synuclein was then dialyzed against 5 L of PBS and stored at −20 ° C.

Dopamine was used to oxidize α-synuclein. An equal volume of 200 uM solution of dopamine-HCL (Sigma P5244) prepared in 10 mM PBS, pH 7.4 and a 28 μM solution of α-synuclein (Rpeptide) in 10 mM PBS, pH 7.4 were combined. The resulting 14 uM α-synuclein / 100 uM dopamine was incubated at 37 ° C. for O / N (overnight). The oxidized α-synuclein was then dialyzed against PBS and stored at −20 ° C.

Various natural and chimeric forms of synuclein proteins have been produced to screen a diverse library of anti-α-synuclein antibodies. Screening constructs included: human, mouse, rat and cynomolgus monkey α-synuclein, human β-synuclein, human γ-synuclein (FIGS. 21 and 22) and finally, residues 120- of α-synuclein. An α-synuclein derivative lacking 140. In addition, a series of four shuffle constructs: A-Syn-AAKK-BAP, A-Syn-BAAK-BAP, A-Syn-BBAA-BAP, A-Syn-BBKK-BAP (SEQ ID NOs: 11-14). ) Was produced. These constructs contained a linear stretch of human α-synuclein (A), human β-synuclein (B) and chicken α-synuclein (K). Genes containing a biotin receptor peptide (BAP) tag fused to the C-terminus of the ligand were cloned to promote each site-specific biotinlation of the ligand. Biotinylation allowed binding of the ligand to the beads used in the soluble ELISA format. Mammalian expression vectors carrying various α-synuclein BAP tag fusion constructs (ASynBAP) were constructed. The ligand was expressed in HEK 293 cells using transient transfection (Genmab A / S).

2. 2. The immunized antibody HuMab-sinucrane was double knocked out of the mouse immunoglobulin (Ig) heavy chain and the mouse κ light chain to completely prevent the expression of the antibody in mice, HuMAb mouse strains HCo17-BALB / c and HCo12-. Obtained from immunization of BALB / c mice (human monoclonal antibody; Medarex Inc., San Jose, CA, USA). Various mouse strains are transgeniced by insertion of human Ig heavy chain and human Ig κ light chain loci, which have a number of human VH (heavy chain variable region) and VL (light chain variable region) genes. different.

Forty-eight mice were alternately immunized intraperitoneally (IP) with 20 μg of antigen and subcutaneously (SC, caudal basal) with the same immunogen at intervals of 14 days. Up to 8 immunizations were performed with 4 IPs and 4 SCs.

The first immunization was performed on an α-synuclein immunogen in a complete Freund's adjuvant (CFA; Defco Laboratories, Detroit, MI, USA) and the next immunization was performed in an incomplete Freund's adjuvant (IFA). Serum antibody titers were found to be sufficient (1/50 or less serum dilutions were positive in the antigen-specific screening assay described herein above in biweekly screening events at least twice in a row. When (found), mice were given (IV) two additional doses of 10 μg α-synuclein immunogen. In 100 μL PBS 4 and 3 days prior to fusion.

The immunization protocol is shown in FIG.

Antibody 37 was obtained from an immunization protocol using human full-length α-synuclein-fibrils, alternating with α-synuclein C-terminally truncated forms containing amino acids 1-60 and 1-119.

Antibody 285 was obtained from an immunization protocol that used human α-synuclein-monomers 1-140 for the first four immunizations. In the absence of antibody titer, immunization was continued with fibrils (ip / sc) or with monomers.

3. 3. HuMAb mice with sufficient antigen-specific antibody titer expression defined on HuMab hybridoma production were slaughtered and the spleen and lymph nodes adjacent to the abdominal aorta and vena cava were harvested. Fusion of splenic cells and lymph node cells with a mouse myeloma cell line is performed by electrical fusion using the CEEF 50 Electrofusion System (CytoPulse Sciences, Glen Burnie, MD, USA), basically according to the manufacturer's instructions. rice field. The fusion cells were subjected to 10% Fetal Clone I Bovine serum (Perbio) in HyQ mADCF-Mab (Perbio), 1 mM sodium pyruvate (Cambrex), 0.5 U / mL penicillin, 0.5 U / mL streptomycin (Perbio). Fusion containing Cambrex), 50 μM 2-mercaptoethanol (Invitrogen), 600 ng / mL interleukin 6 (IL-6) (Strathmann), 1 × HAT (Sigma) and 0.5 mg / mL Invitrogen. It was sown in the medium. After 10 days, supernatants were collected and cells were subjected to 10% Fetal Clone I Bouvine serum in HyQ mADCF-Mab, 0.5 U / mL penicillin, 0.5 U / mL streptomycin, 600 ng / mL IL-6. And washed with collection medium containing 1 × proHT (Cambrex). The supernatant of the hybridoma culture was screened by a primary screening assay. The supernatant was characterized for binding to eight different ligands. These include four orthologs: human, mouse, rat and cynomolgus monkey, human α-synuclein β-synuclein and human γ-synuclein (SEQ ID NOs: 37-41), and finally they are the residue 120 of α-synuclein. Their ability to bind to human α-synuclein derivatives lacking ~ 140 was tested.

Screening for anti-α-synuclein antibodies was performed using a high throughput suspension ELISA format using an automated liquid handling system (Genmab A / S). Plate readings were performed by two systems, 384-well plates were read using an Applied Biosystems FMAT 8200, and 1536-well plates were read using a Molecular Devices ImageXpress Velos CYTOMETER.

In the primary screening, clones were characterized by their ability to bind to eight different ligands. These are a series of four shuffle constructs: A-Syn-AAKK-BAP, A-Syn-BAAK-BAP, A-Syn-BBAA-BAP, A-Syn-BBKK-BAP (SEQ ID NOs: 11-14), α. -Contains 120-140 deletion-BAP, nitrated human α-synuclein-BAP and oxidized human α-synuclein-BAP.

Briefly, serum or supernatants potentially containing α-synuclein-specific antibodies can be added to the beads to allow binding to α-synuclein and / or constructs derived from α-synuclein. Binding of anti-α-synuclein antibody is detected using a fluorescent conjugate, DyLight649 conjugated goat anti-human IgG (Fc-specific). Two known mouse anti-α-synuclein antibodies, LB509 and Syn211 were included in the screening as positive controls. To ensure specific detection of α-synuclein antibody, anti-α-synuclein serum pool is used as a negative control in 384-well format antibody titer screening, while human ChromPure IgG is added to 1536-well format 8-beads. Used in based assays.

Hybridoma cells from the best primary wells were seeded in semi-solid medium prepared from 40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ2 x complete medium (Hyclone, Waltham, USA). For each primary well, wells of Genetix black 6-well plates were sown. Twenty-five subclones were taken from each well using the ClonePix system (Genetix). Subclones were collected in the collection medium. After 7 days, the supernatant of the subclone was screened again for synuclein-specific human IgG binding and human IgG concentrations were measured using Octet (Fortebio, Menlo Park, USA). From each primary well, the best subclone was selected and expanded medium containing only 600 ng / mL IL-6, 0.5 U / mL penicillin, 0.5 U / mL streptomycin and 1 × proHT. ) Expanded inside. This subclone was expanded from one 96-well plate well to one 24-well plate well, to four 24-well plate wells, and to six 6-well plate wells. The clones obtained by this process were referred to as primary clones (PCs).

Further antibody binding studies were performed using Octet 384RED (Fortebio, Menlo Park, USA). A 2 μg / ml HuMab antibody solution was prepared by dilution in a sample diluent (ForteBio, art. No. 18-5028). An amine reactivity sensor (ForteBio, art.no. 18-0008) was used for the immobilization of HuMab. HuMab was diluted in MES pH 6.0 buffer (18-5027) prior to coupling to the amine reactivity sensor. Coupling was performed at 30 ° C. and 1000 rpm as follows: the amine reactivity sensor was pre-moistened in PBS followed by EDC / NHS (ForteBio.) For 300 seconds (as directed by the manufacturer). Art. No. 18-1033 / 18-1034) Activated with an activation solution. The activation sensor was immobilized on HuMab for 600 seconds.

The binding of 37 and 285 in Octet to recombinant human, cynomolgus monkey and mouse α-synuclein, and the lack of binding to human β or γ-synuclein are shown in FIG.

4. Sequence analysis of synuclein-specific HuMab variable regions and cloning in expression vectors Following the manufacturer's instructions, total RNA was prepared from 0.2-5 x 106 hybridoma cells using the SMART RACE cDNA Amplification Kit (Clontech). 5'-RACE-complementary DNA (cDNA) was prepared from 100 ng of total RNA. VH and VL coding regions were amplified by PCR and p33G1f and p33κ expression vectors by ligation-independent cloning (Aslanidis, C. and P.J. de Jong, Nucleic Acids Res 1990; 18 (20): 6069-74). Cloned directly in the frame (including human IgG1 / κ constant region coding sequence). For each antibody, 16 VL clones and 16 VH clones were sequenced. Clone with a suitable open reading frame (ORF) was selected for further research and expression. All combinations of heavy and light chain vectors were transiently co-expressed in FreestyleTM 293-F cells using 293fectin.

For GM37, sequencing the VH region identified an extra cysteine in the CDR3 region at position 106. Cysteine was mutated to serine at position 106 to estimate the possibility of misfolding and disulfide bond formation to reduce antibody activity.

Comparative antibody 9E4 was made based on VH and VL sequences derived from hybridoma PTA-8221 (US Patent Application Publication No. 20080175838) (SEQ ID NOs: 42 and 43).

5. Expression / Purification of Antibodies Antibodies were produced by transfection in HEK2936E cells using the pTT5 vector and PEIpro (National Research Council of Canda) as transient transfection agents. Briefly, heavy and light chains were transfected into HEK293 cells using PEIpro (VWR) and 24 hours after transfection, the cells were supplemented with TN1 (Sigma). Cells were grown until viability approached 50% and antibody yield was measured by easy IgG title (Thermo). The supernatant of the culture was filtered through a 0.2 μm dead-end filter, loaded onto a 5 mL protein A column (rProtein A FF, Amersham Bioscience) and eluted with 0.1 M citrate-NaOH, pH 3. The eluate was immediately neutralized with 2M Tris-HCl, pH 9 and dialyzed overnight (O / N) against 12.6 mM NaH ₂ PO ₄ , 140 mM NaCl, pH 7.4 (B. Braun). .. After dialysis, the sample was sterile filtered on a 0.2 μm dead-end filter. Purity was determined by SDS-PAGE and concentration was measured by turbidimetry and absorbance at 280 nm. The purified antibody was divided and stored at −80 ° C.

Example 2: Evaluation of antibody characterization using surface plasmon resonance Real-time binding of the antibody to α-synuclein was measured using BIAcore® 3000. The capture surface was placed in a first flow cell (Fc1) and a second flow cell (Fc2) of a CM5 chip (BIAcore®) with a polyclonal rabbit anti-mouse antibody (Mouse Antibody Capture Kit (GE Healthcare, Cat.no:): It was prepared by amine-coupling a portion of BR-1008-38). Mouse antibodies were captured in Fc2 at the concentration required to achieve ligand levels of approximately 500 RU. The baseline was stabilized for 10 minutes before injecting the sample (ASynBAP) into Fc 1-2 at 30 μl / min. ASynBAP was tested at 100-3200 nM and 25-3200 RU, respectively. The highest concentration in each series of titrations was tested in duplicate. At the end of each cycle, the surface was regenerated with 10 mM glycine-HCl, pH 1.7 (30 seconds infusion) to remove captured mouse antibodies and specimens. HBS-EP (GE Healthcare, Cat. No: BR-1001-88) was used as test buffer and sample diluent in all experiments and the assay was performed at 25 ° C. All samples were kept at 4 ° C. before collection.

The response value recorded in Fc1 where the capture antibody was fixed but the α-synuclein antibody was not captured was subtracted from the response value in Fc2. A 1: 1 or 2: 1 coupling algorithm was adapted to the dataset using BIA evolution software version 4.1.1. Results are seen in FIGS. 3, 4 and 5, which show the binding of antibodies 37, 285 and 9E4 to human α-synuclein.

Example 3: Epitope Mapping Epitope mapping of antibodies to α-synuclein was performed using a series of overlapping linear peptides in Pepscan (Pepscan Zuidersluisweg 2 8243 RC Lelystad, the Netherlands). Antibody binding to each of the synthesized 20-mer peptides was tested in a Pepscan-based ELISA. A linear peptide array covering the entire coding sequence of α-synuclein, as well as all peptides containing methionine oxide or nitrosylated tyrosine, were incubated with the primary antibody solution (overnight at 4 ° C.). After washing, the peptide array was incubated with antibody peroxidase conjugate (SBA, cat.nr. 2010-05) at a dilution of 1/1000 for 1 hour at 25 ° C. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzothiazolin sulfonate (ABTS) and 2 μl / ml 3 percent H2O2 were added. After 1 hour, the color development was measured. Color development was quantified using a charge-coupled device (CCD) -camera and image processing system. For data processing, values from a CCD camera range of 0-3000 mAU were obtained, similar to a standard 96-well plate ELISA reader. The results were quantified and stored in the Peplab database. From time to time, wells contain bubbles that result in false positive values, the card is manually inspected and the value generated by the bubbles is scored as 0. Binding data for antibodies 37 and 285 to peptides containing the sequence ILEDMP or ILED, respectively, can be seen in FIG.

Example 4: Immunoprecipitation of α-synuclein from human brain homogenates of zonal cortex from patients with Lewy body dementia Coarse zonal cortex from human DLB or healthy controls (marked with ^* ) The ability of the antibody to bind and destroy α-synuclein from the homogenate was analyzed by immunoprecipitation. Frozen samples from the human band cortex (obtained from Tissue Solutions Ltd, Scottland) are cleaved in a cryopreservation device and 100 mg of the sample is 1600 μl cellytic containing a protease inhibitor and a phosphatase inhibitor (Roche). It was added to an M cell thawing reagent (Sigma C2978). Brain tissue was homogenized at 5000 rpm for 4 x 30 seconds using a Precellys bead homogenizer (Bertin technologies, France) until the sample was completely lysed. The solution was centrifuged at 3000 xg and the supernatant was used as a crude homogenate for immunoprecipitation.

For immunoprecipitation, 10 μg of antibody was mixed with magnetic Dynabeads protein G beads using the manufacturer's instructions (Life Technologies, Paisley, UK). Crude brain homogenate was diluted 30-fold in lysis buffer (Sigma). The antibody-bound dynabeads were mixed with 500 ul of diluted homogenate and incubated for 90 minutes at room temperature with continuous mixing in a rotator. After incubation, the beads were washed in wash buffer and the bound antigen was eluted with a non-denaturing elution buffer according to the manufacturer's instructions (Dynabeads G protocol, Life Technologies, Paisley UK). Yields of immunoprecipitation were visualized by Western blotting using a detected mouse monoclonal anti-human α-synuclein antibody (4B12, Thermo Scientific). The band pattern representing the various molecular weight morphologies of the disrupted α-synuclein is that the 37, 37v2 and 285 antibodies are the major α-synuclein species, full length α-synuclein (FL asyn 1-140) and C-terminal. Antibodies 9E4 are 37, 37 variants 2 and 285 antibodies in that cleavage species 1-119 / 122 cannot be immunoprecipitated, while cleavage species (1-135 and 1-119 / 122) can be immunoprecipitated. And the comparative antibody 9E4 (FIG. 9).

Example 5: Inhibition of protease cleavage of α-synuclein fibrils by antibody in cell culture Recombinant α-synuclein monomers and fibrils can be taken up by primary neurons in culture. After uptake of α-synuclein into neurons, as schematically shown in FIG. 10, it can be processed by intracellular proteases such as calpain I at the major protease sensitive sites at amino acids 119/122. To investigate the cleavage of α-synuclein by proteases, mouse primary cortical neurons were subjected to Elvang et al. Prepared as described in 2009 (Elvang et al. J Neurochem. 2009; 110 (5): 1377-87) and in DIV3 (3 days in vitro) to inhibit astrocyte proliferation. , Treated with cytarabine. At DIV4 (4 days in vitro), the neurons were sonicated to a final concentration of 0.7 μM (in a cuphorn sonicator at 50% power for 5 minutes), a preformed α-synuclein source. Treated with fibrils (PFF) alone or with the indicated concentrations of antibody. After 24 hours of incubation, medium was collected and cells were lysed. Western blots were performed on both medium and cytolysate using 4B12 antibody (Figure 11A) (Pierce MA1-90346) and secondary anti-mouse antibody. After probing with 4B12 + anti-mouse, the blot was stripped and probed again with anti-human-IgG antibody. Blots on 4B12 show that there is a strong band at 14 and 12 kDa in medium from cells treated with PFF alone, where 14 kDa represents full length α-synuclein (FL-asyn). 12 kDa represents the C-terminal cleavage fragment 1-119 / 122 (CT-asyn). In addition, there were bands with higher molecular weights that were most likely to represent SDS-resistant oligomeric species. Combination treatment with the isotype control antibody B12 did not alter this pattern of proteolysis or uptake.

In medium from cells treated with fibrils together with 37, there were predominantly full-length α-synuclein (14 kD) and a very small amount of truncated band (12 kD). In cytolysates from cells treated with fibrils together with 37, there is only full-length α-synuclein, indicating that 37 prevents cleavage of FL-α-synuclein. In addition, the total amount of FL-α-synuclein is reduced for cells treated with PFF alone or with B12 control antibody. Several groups (Games et al, Am Jof Pathol, Vol. 182, No. 3, March, 2013; Richie et al, Health, Vol. 4, Special Issue, 1167-1177, 2012; Missizen-Eberz, Bisch. , 2005, 44, 7818-7829; Dufty et al, Am Jof Pathol, Vol. 170, No. 5, May 2007) showed that α-synuclein could be cleaved by calpain-1. The cleavage site for calpain-1 for fibrilyzed α-synuclein was found to be in regions 114-122 (Mishizen-Eberz, Joof Neurochem, 86, 836-847, 2003). In vivo, in transgenic animal and human brains, 1-119 / 122 appears to be the major cleavage product in α-synuclein, and cleavage may be after asparte 119 or asparagine 122. Highly, it is deamidated to aspartate and cleaved by calpain or another protease with similar cleavage specificity. These results indicate that antibody 37 can inhibit C-terminal cleavage of α-synuclein. Since the epitope of antibody 37 overlaps the enzyme calpain-1 binding site, binding of 37 to α-synuclein can directly inhibit calpain-1 -mediated binding and cleavage (FIGS. 10 and 11).

It can also be predicted that 285 epitopes will overlap 37 epitopes and inhibit protease cleavage. Since the amino acid sequence of 37v2 is only different for 37 in one amino acid in CDR and has similar binding to 37, it would be expected that 37v2 would inhibit protease cleavage as well as 37. To investigate whether the effect of the antibody was dose-dependent, a 24-hour experiment was set up with simultaneous addition of PFF and antibody to primary cortical neurons. The concentration of PFF was stable (10 μg / ml), but the concentrations of control antibody B12 and antibodies 37, 37v2 and 285 tested were 10, 5, 1 and 0.1 ug / ml. Α-synuclein on Western blots was detected using a 1904 / 4B12 antibody (Abcam), which binds to both FL and C-terminal cleaved α-synuclein due to its epitope within regions 103-108. (FIG. 11B). As can be seen from FIG. 11B, GM37, 37v2 and GM285 all have dose-dependent inhibition of protease cleavage and near complete inhibition of cleavage at high concentrations of antibody.

Example 6: Antibody-mediated inhibition of seeding of α-synuclein aggregation in cell cultures Some studies have shown that external addition of recombinant α-synuclein fibrillar aggregates enters cells and is endogenous. It has been shown that α-synuclein can be recruited to induce LB-like in vitro and in vitro α-synuclein aggregation and phosphorylation (Volpicelli-Daley et al. 2011, Luk et al. 2012a, Luk et al. 2012b, Recenses et al. 2013, Peelarts et al. 2015). To investigate the seeding of endogenous mouse α-synuclein with recombinant α-synuclein seeds, mouse primary cortical neurons prepared as described above were plate-cultured in 96-well plates (15,000 cells per well). Will be done. On day 5 (DIV) of in vitro culture, 50% of the medium is replaced and supplemented with cytosine arabinoside (final concentration of 1uM). On day 6 of in vitro culture (DIV6), half of the medium is replaced with glial-conditioned medium containing either α-synuclein fibril material, crude fiber seeds or pure seeds. Crude fiber seeds are made from recombinant monomeric human α-synuclein isolated from bacteria, the monomers are filtered through an Amicon Ultra 100.000 cutoff filter (Millipore cat. No UFC51096), PBS, pH 7. The concentration was adjusted to 1 mg / ml in 4. To make fibril crude seeds, the monomer solution was incubated in a thermomixer at 37C with continuous mixing (800 rpm) until a plateau was reached (evaluated by daily measurements with Thioflavin S). To minimize evaporation, a drop of mineral oil was added to cover the solution. The total incubation time is 5-7 days and pure seeds are made from the purified crude fibril seeds by centrifugation and the aggregated pellets are resuspended in fresh PBS. , Sonicate. The antibody is added once with DIV6 with α-synuclein crude seed. Half of the medium in the primary neurons is replaced weekly with glial-conditioned medium and maintained until DIV21. The neurons were immobilized and stained for phospho-synuclein with a rabbit antibody specific for the phosphorylation of α-synuclein at amino acid S129 (abcam 51253), followed by a fluorescently labeled anti-rabbit antibody, and fluorescence was electrophoresed under automatic fluorescence microscopy. Quantify using a Cellomics Arrayscan. Nuclei are detected within one channel and the number of effective cells is defined. Phosphorylated α-synuclein spots were detected within another channel in a predetermined ring-forming region around the nucleus representing the cytoplasm of the cell. The average number of spots per cell was calculated. An example of cell staining is shown in FIG. 12A. Phosphorylated alpha-synuclein spots are not found in untreated neurons. Neurons incubated with crude or pure seeds (1-10 ng per well) induce phosphorylation of α-synuclein (FIG. 12A). In neurites, phosphorylated synucleins appear as spots or spots, and some of the phospho-synucleins within the neurites appear elongated.

For fractionation tests, cells were collected in phosphate buffered saline (PBS) and centrifuged. The pellet was resuspended in 1% triton buffer containing a protease inhibitor. The sample was held on ice for 15 minutes and then sonicated. Samples were centrifuged at 100,000 xg over 30 minutes at 4C. The supernatant was collected and displayed as a soluble fraction. The pellet was washed once in triton buffer, resuspended in 1% SDS buffer and then sonicated. The sample was centrifuged at 100,000 xg for 30 minutes. The supernatant is collected as an insoluble fraction. Protein concentrations were measured, samples were tested on 4-12% SDS_PAGE gels, blotted on membranes, and α-synuclein and phosphorylated α-synuclein (S129P) were each 4B12 / 1904 antibody (Thermoscientific: MA1-90346). -Human synuclein), S129P-asyn antibody (abcam 51253) and mouse synuclein antibody (cell signaling-D37A6).

FIG. 12B shows Western blots of soluble and insoluble fractions from primary neurons with and without crude seeds. As can be seen from FIG. 12B, the addition of seeds results in the accumulation of multimers of endogenous mouse α-synuclein and p-S129-α-synuclein and phosphorylated mouse α-synuclein within the insoluble fraction of the cells.

To test whether the antibody could inhibit seeding, α-synuclein synuclein seeds were used at a concentration of 6.6 nM (10 ng / well). Different concentrations of antibody and α-synuclein seeds were added together at DIV6 to form a dose response (starting from the highest antibody concentration of 133 nM to 133 pM). Neurons were re-fixed, stained for phospho-synuclein (abcam 51253), and fluorescence from cells was quantified using automatic fluorescence microscopy, Cellomics arrayscan. Spots / spots per cell were counted in Cellomics arrayscan. As can be seen from FIG. 12C, both antibodies 37, 37v2 and antibody 285 dose-dependently reduced α-synuclein phosphorylation in neurons with similar maximal inhibition rates (about 70-75%) for 37, 37v2 and 285. ) And had an EC50 of about 5 nM. Separation of cellular proteins into soluble and insoluble fractions after treatment with the antibody at the highest concentration (133 nM), as can be seen from FIG. 12D, all of the antibodies 37, 37v2 and 285 are cleaved and C-terminal to the recombinant crude seed. It is shown that the accumulation of cleaved fragments (CT a-sin) was inhibited, and the accumulation of phosphorylated endogenous mouse α-synuclein in the insoluble fraction and the aggregated form of mouse α-synuclein were reduced.

Example 7. Acute electrophysiological effects of α-synuclein antibody in vivo High expression levels of human α-synuclein are models of overexpression of wild-type α-synuclein under the control of the mouse α-synuclein promoter, the F28-snca transformer. It is present in the hippocampus of genetic mice (Westerlund M, et al. Mol Cell Neurosci. 2008 Dec; 39 (4): 586-91). Evaluation of synaptic transmission and plasticity in the CA1 region of the hippocampus in 4- to 6-month-old male F28-snca transgenic mice and age-matched control mice was performed electrophysiologically in vivo. The data show that basal synaptic transmission was significantly reduced in F28-snca transgenic mice compared to age-matched control mice (FIG. 13).

4- to 6-month-old F28-snca transgenic mice and age-matched control male mice (CRO breeding, Taconic Europe A / S) were subjected to controlled temperature (22 ± 1.5 ° C.) and humidity conditions (55-). 65%) were housed in a single hut and bred on a 12:12 hour light / dark cycle (lighting turned on at 06:00 h). Food and water were freely available.

Animals were anesthetized with an intraperitoneal (ip) injection of urethane (1.2 g / kg). Next, the mouse was attached to the stereotaxic frame, and the body temperature of the mouse was adjusted to 37.5 ° C. by a heating pad to expose the skull. A platinum wire was placed in the frontal bone to serve as a reference and placed in the hippocampus according to the illustrations of Paxinos and Franclin (Paxinos and Franclin's the Mouse Brain in Stereotactic Coordinates, 4th Edition, 2001) in the hippocampus. And additional holes were drilled for insertion of stimulating electrodes: recording, 1.5-1.7 mm posterior to bregma, 1.0-1.2 mm lateral to the median plane, 1.4-1. Of the surface of the brain. 7 mm below; stimulus, 1.8-2.0 mm posterior to bregma, 1.5-1.7 mm lateral to the median plane, 1.5-1.7 mm below the surface of the brain. Animals were left in the stereotaxic frame for the entire duration of the recording and their levels of anesthesia were checked regularly.

The electric field potential (fEPSP) was induced in CA1 by electrical stimulation of the Schaffer side branch every 30 seconds and the depth of the recording electrode was adjusted until the negative fEPSP was recorded in response to a unipolar square pulse. The induced slope of fEPSP was measured at 30-70% of the maximum amplitude of fEPSP.

Once optimal fEPSP was evoked, basal synaptic transmission was assessed by the relationship (input / output relationship) between stimulus intensity and induced fEPSP tilt. Stimulations of various intensities are 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 μA, which are added sequentially in ascending order and repeated 2-3 times at each intensity. Basis synaptic transmission was found to be significantly reduced in F28-snca transgenic mice compared to age-matched control mice.

GM37, GM285 and comparative h9E4 were tested for their ability to block alpha-synuclein-mediated effects using confirmed disorders of basal synaptic transmission in F28-snca transgenic mice.

Recordings were made in all experiments 3-6 hours after administration of a single dose of antibody at a dose of 15 mg / kg (intraperitoneal (ip)). Basis synaptic transmission was recorded in both hippocampi of each animal, if possible, and recorded as individual experiments.

Acute treatment with h9E4 induced a significant improvement in impaired basal synaptic transmission in F28-snca transgenic mice (Tg-snca + h9E4 vs. Tg-snca + PBS, p = 0.002, FIG. 14). However, the improvement with h9E4 was only partial, as indicated by a significant difference from basal synaptic transmission in PBS-treated littermates (p = 0.007).

Acute treatment with GM37 induced a significant improvement in impaired basal synaptic transmission in F28-snca transgenic mice (Tg-snca + GM37 vs. Tg-snca + PBS, p = 0.004, FIG. 15). Basis synaptic transmission in GM37-treated transgenic mice is not very different from basal synaptic transmission in PBS-treated littermates, indicating a complete improvement in the disorder (FIG. 15).

GM285 also induced a significant improvement in impaired basal synaptic transmission in F28-snca transgenic mice (FIG. 16). Basis synaptic transmission in GM285-treated transgenic mice is not very different from basal synaptic transmission in PBS-treated littermates, indicating a complete improvement in the disorder.

Example 8. Microdialysis for assessing human α-synuclein in the brains of awake free-behavior animals A push-pull microdialysis method was used to assess levels of human α-synuclein in interstitial fluid (ISF). Mice were housed in a single hut at controlled temperature (22 ± 1.5 ° C.) and humidity conditions (55-65%) and turned on at a 12:12 hour light / dark cycle (06: 00h). It was bred in). Food and water were freely available. This test was performed in the hippocampus of F28-snca transgenic mice (50-54 weeks old). To enable microdialysis in the hippocampus, mice were anesthetized with isoflurane, an intracerebral guide cannula (CMA) was stereotactically implanted in the brain, and microdialysis probes were placed in the hippocampus according to the illustrations of Paxinos and Franclin 2001. Positioned at (probe tip placement: 3.1 mm posterior and 2.8 mm lateral to the bregma, and 1.3 mm relative to the dura mater). Fixing screws and acrylic cement were used to secure the guide cannula. After implantation of the cannula, mice were recovered from surgery for 2-3 days prior to dialysis.

On the day of the experiment, a 2 mm 1000 kDa cutoff CMA probe was inserted into the guide cannula. The probe was connected to a microdialysis peristaltic pump (MAB20; Microbiotech) with two channels and operated in push-pull mode. The inlet tube of the microdialysis probe was connected to a peristaltic pump (aCSF; mM unit: 147 NaCl, 2.7KCl, 1.2CaCl ₂ , 0.85MgCl ₂ ) that allowed the probe to perfuse artificial cerebrospinal fluid. A peristaltic pump was further connected to the outlet tube to prevent loss of peristaltic fluid from the probe due to withdrawal of peristaltic fluid from the tube. As a perfusion buffer, 25% bovine albumin fraction V (Sigma) was diluted to 0.2% with artificial CSF on the day of use and filtered through a 0.1 μm membrane. The actual flow rate of the pump was measured without the probe connected. Sample tubes were weighed before and after sampling over a given period and flow rates were calculated. Next, the pump was set to a constant flow rate of 1 μL / min. A 120-minute sampling scheme was used throughout the experimental period. The experimental period was set 14-48 hours after probe implantation to avoid tissue damage interference. 14-16 hours after the start of the experiment, GM37, human 9E4 or isotype control (anti-HEL) was injected intraperitoneally (ip) at 15 mg / kg and 6 additional samples (12 hour collection) were injected. Collected. The dialysate was stored at −80 ° C. The concentration of human α-synuclein was determined by ELISA (Covance ELISA kit).

The average of 2 to 3 basal values (4 to 6 hours) prior to antibody treatment was taken as the baseline and set to 100% for each animal. To assess the statistical relevance, the data were evaluated using a two-way ANOVA with repeated measurements. The basal level of human α-synuclein in the hippocampus was 8.1 ± 1.1 ng / ml (mean ± SEM, n = 25, uncorrected for in vitro dialysis probe recovery). Administration of GM37 induced a significant reduction in human α-synuclein in the hippocampus of F28 mice compared to both the comparative antibody, human 9E4, and isotype control (anti-HEL) (FIG. 17).

Example 9: Chronic effect of α-synuclein antibody in vivo. Antibody GM37 improves motor phenotype in a rat Parkinson's disease model.
Targeted overexpression of human α-synuclein to dopaminergic neurons in the rat midbrain is feasible using a recombinant adeno-associated virus vector (rAAV), which is a dopaminergic cell in the substantia nigra. It is associated with progressive loss as well as motor impairment.

AAV2 / 5 serotype adeno-associated virus, which contains chicken β-actinpromotor along with enhancer elements from cytomegalovirus promoters, using adult female Sprague dolly rats (225-250 g), as previously described. Subsequently, human α-synuclein cDNA and WPRE elements were injected to express human α-synuclein in the substantia nigra (SN) (Xu L, Day T, Gao C, Flotte TR, Song S, Virus BJ, Sands). MS, Ponder KP (2001). In this model, human α-synuclein expression has been shown to lead to neurodegeneration of dopaminergic neurons (Maingay M, et al. CNS Specter. 2005 Mar; 10 (3). ): 235-44). To test the efficacy of α-synuclein therapeutic antibodies in this model, antibody treatment was started 2-4 days prior to virus injection and continued until the end of the study (FIG. 18). PBS administration in volume (5 ml / kg: IP) was used as a control. GM37 was administered twice a week at a dose of 15 mg / kg (IP). Human wild-type α-synuclein gene or green fluorescent protein ( GFP) -containing virus particles (rAAV2 / 5) were injected into one side of the SN. Animals were injected 2.0 ml / kg subcutaneously (sc) with Hypnorm® and Dormicum®. The animals were anesthetized with the combination of and placed in a stereotactic frame. The body temperature of the animal was adjusted to 37.5 ° C. with a heating pad to expose the skull. According to the illustrations of Paxinos and Watson (Paxinos & Watson, 1998), Perforated on the right SN in the arrangement of: 5.5 mm posterior and 2.0 mm lateral to Bregma. Single injection of 3 μL rAAV2 / 5-α-syn or rAAV2 / 5-GFP, stereotactic injector. Using a Hamilton syringe coupled to (stereotactic injector), performed at a depth of 7.2 mm below the substantia nigra and at a flow rate of 0.2 μL / min to allow diffusion of the vector in the SN. After the surgery, the animal was returned to the home cage and placed in a heated environment, where the animal was recovered from anesthesia. Motion asymmetry in the cylinder test. Sexual test Trials were evaluated before AAV infusion and 3, 7 and 10 weeks after AAV infusion. The data presented correspond to the ratio of right forefoot use compared to full use of both left and right forefoot. The movement of each animal in the cylinder was photographed for a total of 5 minutes, and manual scoring of the number of contacts using the left and right forefoot over 5 minutes was performed on the final day of the test 10 weeks after virus infusion. At 10 weeks there is significant impairment in AAV-syn compared to AAV-GFP infused rats (p = 0.012). The behavior of GM37-treated animals was different from that of GFP rats (p = 0.163 and p = 0.407 for gm37, respectively), indicating a tendency for improvement in GM37-treated animals. This finding indicates that antibody GM37 can improve the motor phenotype of Parkinson's disease in this rat model (FIGS. 18 and 19).

Example 10: Chronic effect of α-synuclein antibody in vivo. The antibody GM37 inhibits seeding and phosphorylation of endogenous mouse α-synuclein aggregation.
Injection of fibrils preformed by α-synuclein made from recombinant proteins into the dorsal striatum of wild-type mice mobilizes endogenous mouse α-synuclein and neurons in the cortex, amygdala and substantia nigra. Induces the formation of Ser-129 phosphorylated aggregates inside of (Luk et al. 2012, Science. 2012 Nov 16; 338 (6109): 949-53). A total of 45 mice were used to investigate whether the α-synuclein-specific monoclonal antibody GM37 could reduce the appearance of phosphorylated α-synuclein inclusion body formation induced in α-synuclein fibrils in vivo. .. Mice were administered 30 mg / kg intraperitoneally (ip) with GM37, GM37 15 mg / kg intravenously (iv), or vehicle ip (PBS). One day later, mice were anesthetized and 2 ul of recombinant human α-Syn crude seeds (Example 6) (total 2 μg of crude seeds per animal) prepared as described above were localized in one hemisphere. Infused. An incision is made in the skull by making a hole to inject the crude seed, and a single glass pipette is inserted into the right anterior brain so that the inoculum is directed to the dorsal striatum (+2.6 mm below the dura). (Arrangement: + 0.5 mm in front of Bregma, + 2.0 mm to the side of the median). After recovery, mice were given weekly injections of the antibody intraperitoneally (ip) or intravenously (iv) until killed 45 days. In a group of 15 mice / group, GM37 15 mg / kg intravenously (iv), GM37 30 mg / kg intraperitoneally (ip), or PBS (10 ml / kg) intraperitoneally (ip) weekly. It was administered once.

To measure plasma antibody levels, buccal blood was drawn weekly just prior to the next infusion, i.e. 7 days after the last infusion. Plasma was obtained by rotation of 2000 g and incubated at room temperature for 15 minutes, followed by freezing the supernatant at −20 ° C. CSF samples were taken at the end of the test and frozen at −20 ° C. Plasma and CSF samples were analyzed to determine the concentration of human IgG by MSD. Briefly, mouse anti-human IgG (clone MH16-1 (M1268)) was used for capture, plasma or CSF was incubated in wells, followed by sulfo-TAG goat anti-human (MSD cat no:). R32AJ-1) was used as the detection antibody. Plates were analyzed from electrochemical luminescence by MSD.

Antibody levels in plasma are shown in FIG. 20B, showing a dose-dependent increase in antibody plasma concentration and antibody accumulation in plasma over a 6-week period. Antibody levels in csf are shown in FIG. 20C, indicating that about 0.1% antibody levels in plasma can be measured in csf.

Forty-five days after infusion of α-synuclein fibrotic seeds, mice were anesthetized, PBS transcardiacly perfusion, and then neutral buffered paraformaldehyde (4%). Brains were removed and incubated overnight for post-fixation in neutral buffered paraformaldehyde. Immunohistochemistry was performed by Neuroscience assists on serial sections with a thickness of 45 μM. Briefly, using MultiBrain® technology, up to 25 mouse brains were implanted in 3 blocks together block by block to produce 45 μm thick frozen sections in the coronal plane. And collected in a cup containing the antigen storage solution. Every six sections were stained with an antibody against Ser-129 phosphorylated α-synuclein (anti-α-synuclein (phospho S129) antibody [Psin / 81A] ab184674) to reveal the Ser129 phosphorylated α-synuclein reactive structure. ..

PSyn lesions were quantified by manually counting immunoreactive positive cells from images at 10x magnification from 5-7 sections covering the entire substantia nigra from every 6 sections. Counting was done blindly. Cell counts in the acuminatum and substantia nigra were analyzed by one-way ANOVA followed by Bonferroni t-test, where the effects of the GM37 antibody were compared to PBS treatment.

As can be seen from FIG. 20C, treatment with antibody GM37 significantly reduced the number of intracellular inclusion bodies in the substantia nigra compared to PBS controls with either intraperitoneal (ip) or intravenous (iv) treatment. .. The data indicate that antibody GM37 blocks the entry of extracellular pathological α-synuclein into neurons by blocking its transmission between neurons and / or promoting clearance from the ISF by uptake into microglia. By doing so, it is shown that the therapeutic effect of PD can be given. Since this appearance of inclusion bodies is associated with the loss of dopaminergic neurons and the development of Parkinson's disease-like dyskinesia in animal models, treatment with antibody GM37 is associated with the loss of dopaminergic cells and the development of PD dyskinesia. Can give a therapeutic effect.

Example 11: Manufacturability of GM37 and GM37 Variants Anti-α-synuclein antibodies are cultured in mammalian cells under conditions that mimic the manufacturing conditions used to produce clinical grade materials for use in patients. Produced in a thing. It is well known that proteins thus produced undergo post-translational modifications that can affect both the therapeutic efficacy of the antibody as well as the biophysical properties that affect the stability of the antibody over time. Empirical knowledge confirmed from decades of research has identified a series of post-translational modifications that are known to pose a risk of development of a particular molecule. These post-translational modifications have been shown to correlate with the amino acid chains present in the primary sequences of heavy and light chain proteins. Algorithms have been developed that can identify these sequences and determine the potential risks they pose to the feasibility and expression of therapeutic antibodies.

In silico analysis of the primary sequence of an antibody can be used to avoid molecular risk due to its potential to be developed as a therapeutic agent. In particular, detailed analysis of the VH and VL regions can identify unique amino acids that are thought to be important for molecular activity, which can also pose a potential risk to their stability over time. Sequence-specific deamidation has been identified as a potential risk to protein structure. Protein deamidation occurs on the amide side chains of glutamine or asparagine residues and can convert them to carboxylic acid groups (Lorenzo et al. PLOSone, DOI: 10.1371, Dec. (2015)). Non-enzymatic deamidation at neutral pH occurs earlier for asparagine and therefore may be at higher risk than glutamine. This activity is further influenced by subsequent amino acids in the sequence and can occur at a rate of days or years. The actual result of the deamidating protein needs to be evaluated experimentally to determine the effect of the change on both its stability and activity.

We have identified a site for deamidation within the VH region of GM37. Amino acid residue 54 is asparagine (N) followed by glycine (G) at position 55. N54 has a high risk of spontaneous deamidation. To reduce this risk, we created a series of three variants in which asparagine (N) was replaced with serine (S), glutamine (Q) or histidine (H). All three variants were generated in mammalian cell cultures using transient transfection methods (Example 1.5). All three variants exhibited expression and purification properties similar to GM 37 wt (FIG. 23).

For each of the eight products, 400 ml transient transfection was performed using CHOK1SV GS-KO cells that had been in culture for a minimum of 2 weeks. Cells were subcultured 24 hours prior to transfection. All transfections were performed by electroporation with Gene Pulse XCell (Bio-Rad). For each transfection, live cells were resuspended in pre-warmed CD-CHO medium supplemented with 6 mM L-glutamine until 2.86 × 10 ⁷ cells / ml. 40 μg of each established SGV DNA containing the appropriate heavy and light chains was divided into each cuvette (Bio-Rad, GenePulser cuvette, 0.4 cm gap, 165-2088) and 700 μl of cell suspension. The turbid liquid was added. The cells were electroporated at 300 V, 900 μF. The transfected cells were transferred to a pre-warmed medium in an Erlenmeyer flask, and the contents of the cuvette rinsed twice with the pre-warmed medium were also transferred to the flask. Transfectant cultures were incubated in a shaking incubator at 36.5 ° C., 5% CO ₂ , 85% humidity, 140 rpm for 6 days. Cell viability was measured at harvest using a Cedex HiRes automatic cell counter (Rosche).

To assess the importance of residue 54 in binding to human α-synuclein, we analyzed the ability of the mutant to bind in two different experiments. Using a competitive ELISA format, we evaluated the possible effects of changes at residue 54 on the ability of GM37 to bind α-synuclein in solution. By assessing the concentration of synuclein capable of inhibiting antibody binding to a synuclein-coated ELISA plate, we found that all three variants maintain the same binding as GM 37 wt. It was shown to bind to α-synuclein with high affinity resulting in an IC50 of 1-2 nM (FIG. 24). The following antibodies at a constant concentration (0.3 μg / ml), GM37 (referred to as GM37 wt), GM37 variant 1, GM37 mutation, using human α-synuclein in the range 0-1000 nM over 60 minutes at room temperature. Competitive assays were performed using the respective preincubations of body 2 and GM37 variant 3. The remaining unbound antibody was captured and measured by electrochemiluminescence (MSD, Gathersburg, MD) on an ELISA plate coated with 100 ng / ml recombinant human α-synuclein using an anti-human detection antibody. The IC50s of interaction are 1.9 nM, 1.6 nM, 2.1 nM and 1.4 nM for GM37 wt, GM37 variant 1, GM37 variant 2 and GM37 variant 3, respectively (Prism Graphpad®). When determined using).

Using surface plasmon resonance (SPR), we evaluated the real-time rate of binding of GM 37 wt (2 batches) and 3 variants (Example 2). Human α-synuclein was captured on a slide (ligand) and each antibody was tested at multiple concentrations as a sample. Analysis of the binding curve in the presence of the antibody at multiple concentrations showed and measured that the on rate was the same for all four antibodies, as was the case when the antibody was removed from buffer. Off-rate showed no statistical difference between the antibodies. Using a 1: 1 binding algorithm, all four antibodies have approximately the same binding constant (FIG. 25).

To assess the effect of changes in N54 on the functional activity of GM37, we analyzed the ability of antibodies to block synuclein seeding activity in cultures of primary neurons (Example 6). .. Seeding levels are measured using antibodies specific for phospho-synuclein. All three antibodies were able to block seeding when measured by the phospho-synuclein signal (FIG. 26). Moreover, the level of inhibition was the same for all four antibodies. This cell-based data further confirms binding data that amino acid 54 in the VH region is not required for binding affinity for human α-synuclein or for inhibition of seeding in primary cell-based assays. Furthermore, we have found that all three of these antibodies were capable of production using standard expression and purification methods. Interestingly, one of the mutants N54Q showed an increase in production over the other mutants, which is very important when the antibody is produced on a large scale and commercially. These data support the potential to reduce the potential risk of deamidation by replacing asparagine (N) with another amino acid without concern for reduced efficacy.

Samples of the antibodies wt GM37, var1, var2 and var3 were steadily increased in temperature over time to reduce the level of aggregation to Prometheus.
NT. 48, NanoTemper Technologies) measured simultaneously. Aggregation initiation temperatures were found to be similar for GM37 and GM37-mutants, but the lowest levels of aggregation were observed for GM37-Var2 (FIG. 27).

The present invention may include the following aspects.
[1]
A monoclonal antibody capable of specifically binding to human α-synuclein.
Amino acids 112-117 of human α-synuclein (SEQ ID NO: 10) (SEQ ID NO: 9 (ILE)
An antibody that binds to an epitope in DMP)), or its antigenic binding that binds to the epitope.
Combined fragment.
[2]
The antibody has the light chain variable region and sequence of SEQ ID NO: 8 for binding to the epitope.
It is possible to compete with antibodies that contain heavy chain variable regions numbered 7, 30, 31 or 32.
The monoclonal antibody according to claim 1 or an antigen-binding fragment thereof.
[3]
Amino acids 112-115 (SEQ ID NO: 19 (SEQ ID NO: 19)) of human α-synuclein (SEQ ID NO: 10)
The thing according to claim 1, which is capable of specifically binding to an epitope in ILED).
A clonal antibody, or its antigen-binding fragment that binds to said epitope.
[4]
The antibody exhibits the heavy chain variable region and arrangement of SEQ ID NO: 26 for binding to the epitope.
1 or 3 which is capable of competing with an antibody comprising the light chain variable region of column number 27.
The monoclonal antibody described, or an antigen-binding fragment thereof.
[5]
The monok according to any one of claims 1 to 4, which comprises or comprises an intact antibody.
Ronal antibody, or its antigen-binding fragment.
[6]
The monoclonal antibody is subtype IgG1, IgG2, IgG3 or IgG4.
The monoclonal according to any one of claims 1 to 5, which is selected from the group consisting of the antibodies of the above.
An antibody, or an antigen-binding fragment thereof.
[7]
Fv fragments (eg single chain Fv and disulfide bond Fv), Fab-like flags
Mento (eg Fab fragment, Fab'fragment and F (ab) 2 flagme
And domain antibodies (eg, a single VH variable region or VL variable region)
Any of claims 1-6 comprising or consisting of an antigen-binding fragment selected from
The monoclonal antibody according to one item thereof, or an antigen-binding fragment thereof.
[8]
The antibody or antigen binding fragment has the following characteristics:
a) For 0.5-10 nM α-synuclein, such as 1-5 nM or 1-2 nM
Binding affinity (KD);
b) Ability to inhibit protease cleavage of α-synuclein fibrils;
c) Improves impaired basal synaptic transmission in F28-snca transgenic mice
Ability to do good;
d) Relax of α-synuclein in mouse hippocampus as measured by in vivo microdialysis
Ability to reduce bells;
e) Restores motor function in a rat model of Parkinson's disease when administered chronically
Ability to make;
f) α-synuclein seeding (in vitro and / or Parkinson's disease)
Ability to prevent the accumulation of insoluble phosphorylated α-synuclein in a mouse model of
/or
g) Ability to bind to cleavage α-synuclein in the human brain
The monoclonal antibody according to any one of claims 1 to 7, which indicates one or more of the above.
Or its antigen-binding fragment.
[9]
The invention according to any one of claims 1 to 8, which is a human, humanized, recombinant or chimeric antibody.
Monoclonal antibody, or its antigen-binding fragment.
[10]
(A) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1;
(B) Heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2;
(C) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3;
(D) Light chain CDR1 having the amino acid sequence of SEQ ID NO: 4;
(E) Light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and
(F) Light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.
A monoclonal antibody comprising, or the model according to any one of claims 1 to 2 and 5 to 9.
Nocronal antibody, or fragment thereof.
[11]
10. The claim 10 comprises the heavy chain variable region of SEQ ID NO: 7 or the light chain variable region of SEQ ID NO: 8.
Monoclonal antibody.
[12]
Containing a heavy chain consisting of the variable region of SEQ ID NO: 7 and a light chain consisting of the variable region of SEQ ID NO: 8.
The monoclonal antibody according to claim 11.
[13]
(A) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1;
(B) Heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 33;
(C) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3;
(D) Light chain CDR1 having the amino acid sequence of SEQ ID NO: 4;
(E) Light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and
(F) Light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.
A monoclonal antibody comprising, or the model according to any one of claims 1 to 2 and 5 to 9.
Nocronal antibody, or fragment thereof.
[14]
13. The thirteenth aspect comprising the heavy chain variable region of SEQ ID NO: 30 or the light chain variable region of SEQ ID NO: 8.
Monoclonal antibody listed.
[15]
Includes a heavy chain consisting of the variable region of SEQ ID NO: 30 and a light chain consisting of the variable region of SEQ ID NO: 8.
The monoclonal antibody according to claim 14.
[16]
(A) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1;
(B) Heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 34;
(C) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3;
(D) Light chain CDR1 having the amino acid sequence of SEQ ID NO: 4;
(E) Light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and
(F) Light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.
A monoclonal antibody comprising, or the model according to any one of claims 1 to 2 and 5 to 9.
Nocronal antibody, or fragment thereof.
[17]
16. The 16th aspect comprising the heavy chain variable region of SEQ ID NO: 31 or the light chain variable region of SEQ ID NO: 8.
Monoclonal antibody listed.
[18]
17. Claim 17 comprising a heavy chain consisting of the variable region of SEQ ID NO: 31 and a variable region of SEQ ID NO: 8.
The monoclonal antibody described in.
[19]
(A) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1;
(B) Heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 35;
(C) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3;
(D) Light chain CDR1 having the amino acid sequence of SEQ ID NO: 4;
(E) Light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and
(F) Light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.
A monoclonal antibody comprising, or the model according to any one of claims 1 to 2 and 5 to 9.
Nocronal antibody, or fragment thereof.
[20]
19. The 19th aspect comprising the heavy chain variable region of SEQ ID NO: 32 or the light chain variable region of SEQ ID NO: 8.
Monoclonal antibody listed.
[21]
20. The heavy chain consisting of the variable region of SEQ ID NO: 32 and the variable region of SEQ ID NO: 8 are included.
The monoclonal antibody described in.
[22]
(A) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 20;
(B) Heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 21;
(C) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 22;
(D) Light chain CDR1 having the amino acid sequence of SEQ ID NO: 23;
(E) Light chain CDR2 having the amino acid sequence of SEQ ID NO: 24; and
(F) Light chain CDR3 having the amino acid sequence of SEQ ID NO: 25
A monoclonal antibody comprising, or the model according to any one of claims 1, 3 and 5-9.
Nocronal antibody, or fragment thereof.
[23]
22. The heavy chain variable region of SEQ ID NO: 26 or the light chain variable region of SEQ ID NO: 27 is included.
The monoclonal antibody described.
[24]
2. Claim 2 comprising a heavy chain consisting of the variable region of SEQ ID NO: 26 and a variable region of SEQ ID NO: 27.
3. The monoclonal antibody according to 3.
[25]
A preparation comprising the monoclonal antibody according to any one of claims 1 to 24, wherein the preparation comprises the above.
The preparation is not capable of binding to α-synuclein or the anti-α- of the preparation.
Synuclein Substantially free of naturally occurring antibodies that do not substantially alter functionality, said function
Gender
(I) Binding affinity of the anti-α-synuclein antibody for α-synuclein (KD);
(Ii) The anti-α-synuclein that inhibits protease cleavage of α-synuclein fibrils.
In-antibody capacity;
(Iii) Impairment of basal synaptic transmission in F28-snca transgenic mice
The ability of the anti-α-synuclein antibody to ameliorate harm;
(Iv) α-synuclein in mouse hippocampus as measured by in vivo microdialysis
The ability of the anti-α-synuclein antibody to reduce the level of
(V) Motor function in a rat model of Parkinson's disease when administered chronically
The ability of the anti-α-synuclein antibody to recover,
(Vi) α-synuclein seeding (in vitro and / or perkinso)
Ability to prevent the accumulation of insoluble phosphorylated α-synuclein in a mouse model of illness;
Or
(Vii) Ability to bind to cleavage α-synuclein in the human brain
A preparation selected from the group consisting of.
[26]
A preparation comprising the monoclonal antibody according to any one of claims 1 to 24, wherein the preparation comprises the above.
The amino acid composition of the monoclonal antibody is compared with the structure of the natural anti-α-synuclein antibody.
It has a structural change in the row, and the structural change causes the monoclonal antibody to become the natural anti-α-.
It exhibits altered functionality compared to the functionality exhibited by synuclein antibodies, and the functionality is said to be
,
(I) Binding parent of the anti-α-synuclein monoclonal antibody to α-synuclein
Harmony (KD);
(Ii) The anti-α-synuclein that inhibits protease cleavage of α-synuclein fibrils.
Capability of in-monoclonal antibody;
(Iii) Impairment of basal synaptic transmission in F28-snca transgenic mice
The ability of the anti-α-synuclein monoclonal antibody to ameliorate harm;
(Iv) α-synuclein in mouse hippocampus as measured by in vivo microdialysis
The ability of the anti-α-synuclein monoclonal antibody to reduce the level of
(V) Motor function in a rat model of Parkinson's disease when administered chronically
Ability of the anti-α-synuclein monoclonal antibody to restore;
(Vi) α-synuclein seeding (in vitro and / or perkinso)
Ability to prevent the accumulation of insoluble phosphorylated α-synuclein in a mouse model of illness;
Or
(Vii) Ability to bind to cleavage α-synuclein in the human brain
A preparation selected from the group consisting of.
[27]
The monoclonal antibody according to any one of claims 1 to 24 or claim 25 to 26.
A pharmaceutical composition comprising the preparation according to any one of the above and a pharmaceutically acceptable carrier.
[28]
A nucleic acid encoding the antibody or fragment according to any one of claims 10 to 24.
[29]
The monoclonal antibody according to any one of claims 1 to 24, which is used for treatment.
Or the antigen-binding fragment thereof, or the antigen-binding fragment according to any one of claims 25 to 26.
Preparation.
[30]
In any one of claims 1-24 for use in treating synucleinopathy
The monoclonal antibody according to the above, or an antigen-binding fragment thereof or claims 25 to 26.
The preparation according to any one of the following items.
[31]
Parkinson's disease, idiopathic and hereditary Parkinson's disease, Gaucher's disease (GD), diffuse
Sexual Lewy Body Disease (DLBD), Lewy Body Dementia with Alzheimer's Disease (LBV), Combined
Cures Alzheimer's disease and Parkinson's disease, pure autonomic failure or multiple system atrophy
The monoclonal antibody according to claim 30, or an antigenic antibody thereof, for use in treating.
Combined fragment.
[32]
The monoclonal according to any one of claims 1 to 24 for use in the manufacture of a drug.
The antibody, or an antigen-binding fragment thereof, or any one of claims 25 to 26.
Its preparation.
[33]
Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gaucher's disease (GD)
, Diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBV),
Combined Alzheimer's disease and Parkinson's disease, pure autonomic failure or multiple system atrophy
32. The agent of claim 32, for use in treating contraction.
[34]
A method of treating Parkinson's disease or other synucleinopathy in a subject.
, The monoclonal antibody according to any one of claims 1 to 24 or an antigen binding flag thereof.
Mento, or the preparation according to claims 25-26 or the pharmaceutical composition according to claim 27.
, A method comprising the step of administering to said subject in an effective amount.
[35]
34. The method of claim 34, wherein the treatment is long-term.
[36]
35. The method of claim 35, wherein the long-term treatment lasts for at least 2 weeks.
[37]
34. The method of claim 34, wherein the subject is a human.
[38]
The antibody according to any one of claims 1 to 24, or an antigen thereof, for use in treatment.
The binding fragment, or the preparation according to any one of claims 25 to 26, or a claim.
Item 27. A kit containing the pharmaceutical composition according to Item 27.
[39]
The monoclonal antibody according to any one of claims 1 to 24, which is detectablely labeled.
Or its antigen binding fragment.
[40]
The detectable label is a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, or the like.
39 is an enzyme-labeled monoclonal antibody, an antigen-binding fragment thereof.
..
[41]
Used to detect or measure the presence or amount of the α-synuclein in the subject's brain
The monoclonal antibody according to any one of claims 39 to 40, the antigen thereof, for use.
Bound fragment, preparation or pharmaceutical composition.
[42]
The detection or measurement of the anti-synuclein antibody bound to the α-synuclein
The monoclonal according to any one of claims 39 to 41, comprising in vivo imaging.
Antibodies, antigen-binding fragments thereof, preparations or pharmaceutical compositions.
[43]
The anti-synuclein antibody to which the detection or measurement was bound to the α-synuclein.
39-4, comprising exviboimaging the antigen-binding fragment thereof.
The monoclonal antibody according to any one of 1 and its antigen-binding fragment, preparation or preparation thereof.
Is a pharmaceutical composition.
[44]
Used in the manufacture of drugs for the treatment, diagnosis or imaging of synucleinopathy
The monoclonal antibody according to any one of claims 1 to 24, or an antigenic antibody thereof.
A combination fragment, or the preparation according to any one of claims 25 to 26, or claim.
The pharmaceutical composition according to 27, or the krona according to any one of claims 39 to 40.
Use of an antibody or antigen-binding fragment thereof, or a preparation or pharmaceutical composition.
[45]
The drug is Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gauci.
Disease (GD), Diffuse Lewy Body Disease (DLBD), Lewy Body Dementias Alzheimer's Disease
(LBV), complex Alzheimer's disease and Parkinson's disease, pure autonomic failure
And the monok of claim 44, for use in the treatment of multiple system atrophy.
Use of Ronal antibody, or antigen-binding fragment thereof, or preparation or pharmaceutical composition
..
[46]
Treat, diagnose or image Parkinson's disease or other synucleinopathy in a subject
The monoclonal antibody according to any one of claims 1 to 24, which is a method for aging.
, Or an antigen-binding fragment thereof, or the key according to any one of claims 25 to 26.
The product, the pharmaceutical composition according to claim 27, or any one of claims 39 to 40.
With a monoclonal antibody, or an antigen-binding fragment thereof, a preparation or a pharmaceutical composition.
A method comprising the step of administering to the subject by efficacy.
[47]
46. The method of claim 46, wherein the treatment is long-term.
[48]
47. The method of claim 47, wherein the long-term treatment lasts for at least 2 weeks.
[49]
48. The method of claim 48, wherein the subject is a human.
[50]
Detecting or measuring the presence or amount of the α-synuclein in a subject's brain or body fluid
The monoclonal antibody according to any one of claims 39 to 40 for use in the above.
Antigen-binding fragment, preparation or pharmaceutical composition.
[51]
The detection or measurement of the anti-synuclein antibody bound to the α-synuclein
The monoclonal antibody according to claim 50, which comprises in vivo imaging, an antigen-binding antibody thereof.
Rugments, preparations or pharmaceutical compositions.
[52]
The anti-synuclein antibody to which the detection or measurement was bound to the α-synuclein.
51.
Monoclonal antibodies listed, antigen-binding fragments thereof, preparations or pharmaceutical compositions.
[53]
Smell of cell lines such as human cell lines, non-human mammalian cell lines, insect, yeast or bacterial cell lines
The antibody according to any one of claims 1 to 24, or an antigen thereof, produced or produced in the art.
Combined fragment.
[54]
CHO cell line, HEK cell line, BHK-21 cell line, mouse cell line (myeloma cell line, etc.)
), Fibrosarcoma cell line, PER. C6 cell line, HKB-11 cell line, CAP cell line and H
The antibody of claim 53, or antigen binding thereof, produced in a uH-7 human cell line.
Fragment.
[55]
Contains the constant region defined in SEQ ID NO: 18 and the κ constant region defined in SEQ ID NO: 17.
, The monoclonal antibody according to any one of claims 1 to 21, the antigen-binding flagmen thereof.
door.
[56]
Contains the constant region defined in SEQ ID NO: 28 and the κ constant region defined in SEQ ID NO: 29.
, The monoclonal antibody according to any one of claims 22 to 24, the antigen-binding flagme thereof.
Nto.

Claims (17)

(A) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1;
(B) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2;
(C) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3;
(D) Light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4;
(E) Light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and (f) Light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.
A monoclonal antibody or antigen-binding fragment thereof capable of specifically binding to human α-synuclein. The monoclonal antibody according to claim 1, which comprises the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8. (A) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1;
(B) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 33;
(C) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3;
(D) Light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4;
(E) Light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and (f) Light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.
A monoclonal antibody or antigen-binding fragment thereof capable of specifically binding to human α-synuclein. The monoclonal antibody according to claim 3, which comprises the heavy chain variable region of SEQ ID NO: 30 and the light chain variable region of SEQ ID NO: 8. (A) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1;
(B) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 34;
(C) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3;
(D) Light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4;
(E) Light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and (f) Light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.
A monoclonal antibody or antigen-binding fragment thereof capable of specifically binding to human α-synuclein. The monoclonal antibody according to claim 5, which comprises the heavy chain variable region of SEQ ID NO: 31 and the light chain variable region of SEQ ID NO: 8. (A) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1;
(B) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 35;
(C) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3;
(D) Light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4;
(E) Light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and (f) Light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.
A monoclonal antibody or antigen-binding fragment thereof capable of specifically binding to human α-synuclein. The monoclonal antibody according to claim 7, which comprises the heavy chain variable region of SEQ ID NO: 32 and the light chain variable region of SEQ ID NO: 8. (A) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 20;
(B) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 21;
(C) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 22;
(D) Light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 23;
(E) Light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 24; and (f) Light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 25.
A monoclonal antibody or antigen-binding fragment thereof capable of specifically binding to human α-synuclein. The monoclonal antibody of claim 9, comprising the heavy chain variable region of SEQ ID NO: 26 and the light chain variable region of SEQ ID NO: 27. The monoclonal antibody according to any one of claims 1 to 10, or an antigen-binding fragment thereof, comprising or comprising an intact antibody. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the monoclonal antibody is selected from the group consisting of antibodies of subtypes IgG1, IgG2, IgG3 or IgG4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, which comprises or comprises an antigen-binding fragment selected from the group consisting of Fv fragments, Fab-like fragments and Fab fragments. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, which is a human , recombinant or chimeric antibody. A nucleic acid encoding the antibody or fragment according to any one of claims 1 to 14. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 for use in treatment. Synucleinopathy, or Parkinson's disease, idiopathic and hereditary Parkinson's disease, Gaucher's disease (GD), diffuse Lewy body disease (DLBD), Lewy body dementias Alzheimer's disease (LBV), combined Alzheimer's disease The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 for use in treating Parkinson's disease, pure autonomic failure or multiple system atrophy.

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